Neural cell extracellular vesicles

ABSTRACT

Disclosed herein are neural extracellular vesicles (EVs) and methods of using these EVs in the treatment of spinal cord injury, stroke, and traumatic brain injury and neurodegenerative disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.16/414,576, filed May 16, 2019; which is a continuation of U.S. patentapplication Ser. No. 15/770,881, filed Apr. 25, 2018; which is a 371National Stage Application of International Application No.PCT/US2016/062245, filed Nov. 16, 2016; which claims the benefit of U.S.Provisional Application No. 62/256,823, filed Nov. 18, 2015. Each of theforegoing applications are hereby incorporated herein by reference intheir entirety.

BACKGROUND

Diseases and injuries to the nervous system, including congenitaldisorders, cancers, degenerative diseases, and spinal cord injury,affect millions of people of all ages. Congenital disorders occur whenthe brain or spinal cord does not form correctly during development.Cancers of the nervous system result from the uncontrolled spread ofaberrant cells. Degenerative diseases occur when the nervous systemloses functioning of nerve cells. There is evidence that damage can bereversed by replacing lost cells with new ones derived from cells thatcan mature into nerve cells, called neural stem cells. However,transplanted stem cells may and often do migrate from the site ofimplantation and undergo some cell transformation that lead to ateratoma formation. In addition, due to their size, stem cells are oftendirectly injected into the CNS which can induce complications. InvasiveCNS surgeries place the patient at risk due to complication that occurduring and after delivery, including but not limited to hemorrhages andedema. Stem cells administered systemically often end up being lodged insmall capillaries which can induce undesired effects in the lung and maynot even reach the disease or injury. Stem cell therapy can also triggeran elicit and adverse immune response.

SUMMARY

Disclosed herein are neural extracellular vesicles (EVs) and methods ofusing these EVs in the treatment of spinal cord injury, stroke, andtraumatic brain injury and neurodegenerative diseases.

The disclosed EVs can be obtained in some embodiments, by culturingneural progenitor (NP) cells that were produced from pluripotent stemcells (e.g. human embryonic stem cells (ESCs) or induced pluripotentstem cells (iPSCs)) in cell culture medium under conditions and for atime sufficient for the NP cells to produce EVs, and isolating said EVsfrom the culture medium. In some embodiments, the NP cells are SOX 1+,SOX 2+, OCT4-, and NESTIN+.

The disclosed EVs can be obtained in some embodiments by culturingneural cells derived directly or indirectly from pluripotent stem cells(PSCs) (e.g. ESCs or iPSCs) in cell culture medium under conditions andfor a time sufficient to produce EVs, and isolating said EVs from theculture medium. In some embodiments, the PSC-derived neural cellcomprises a glial cell, such as an astrocyte or oligodendrocyte. In someembodiments, the PSC-derived neural cell comprises a neuron. In someembodiments, the PSC-derived neural cell are differentiated from NPcells derived from the hES cells. In some embodiments, the PSC-derivedneural cells are differentiated directly from PSCs.

Also disclosed are EVs obtained by culturing astrocytes of any origin incell culture medium under conditions and for a time sufficient for theastrocytes to produce EVs, and isolating said EVs from the culturemedium.

The disclosed EVs can alternatively be obtained in some embodiments, byculturing mesenchymal stem cells (MSCs) that were produced from PSCs incell culture medium under conditions and for a time sufficient for theMSC cells to produce EVs, and isolating said EVs from the culturemedium.

As disclosed herein, EVs produced from hNP cells (also referred toherein as NPEX) had 1653 proteins (see Table 9) that were not identifiedin EVs from stem-cell-derived astrocytes (also referred to herein asAPEX) or mesenchymal stem cells (also referred to herein as MSCEX).Therefore, in some embodiments, the EVs comprises 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600,700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600 or more proteinbiomarkers listed in Table 9.

As disclosed herein, EVs produced from APEX had 596 proteins (see Table8) that were not identified in NPEX or MSCEX. Therefore, in someembodiments, the EVs comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500 or more proteinbiomarkers listed in Table 8.

As disclosed herein, EVs produced from MSCEX had 536 proteins (see Table7) that were not identified in APEX or MSCEX. Therefore, in someembodiments, the EVs comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30,40, 50, 60, 70, 80, 90, 100, 200, 300, 400, or more protein biomarkerslisted in Table 7.

In some embodiments, the disclosed EVs are produced from a substantiallyhomogeneous population of cells. In some embodiments, the disclosed EVsare produced from non-transformed cells.

Also disclosed are compositions containing the disclosed EVs. In someembodiments, the composition comprises the disclosed EVs in abiocompatible scaffold, such as a hydrogel. Suitable hydrogels includetemperature dependent hydrogels that solidify or set at bodytemperature, e.g., PLURONICS™; hydrogels crosslinked by ions, e.g.,sodium alginate; hydrogels set by exposure to either visible orultraviolet light, e.g., polyethylene glycol polylactic acid copolymerswith acrylate end groups; and hydrogels that are set or solidified upona change in pH, e.g., TETRONICS™. The hydrogel can, for example, includeany of the following: polysaccharides, proteins, polyphosphazenes,poly(oxyethylene)-poly(oxypropylene) block polymers,poly(oxyethylene)-poly(oxypropylene) block polymers of ethylene diamine,poly(acrylic acids), poly(methacrylic acids), copolymers of acrylic acidand methacrylic acid, poly(vinyl acetate), and sulfonated polymers.

In some embodiments, the composition comprising the disclosed EVsfurther comprises one more neurotrophic agents. The composition canfurther comprises one or more agents selected from the group consistingof leukemia inhibitory factor (LIF), brain-derived neurotrophic factor(BDNF), ciliary neurotrophic factor (CTNF), epidermal growth factorreceptor (EGF), basic fibroblast growth factor (bFGF), FGF-6,glial-derived neurotrophic factor (GDNF), ciliary neurotrophic factor(CTNF), granulocyte colony-stimulating factor (GCSF), hepatocyte growthfactor (HGF), IFN-γ, insulin-like growth factor binding protein(IGFBP-2), IGFBP-6, IL-1ra, IL-6, IL-8, monocyte chemotactic protein(MCP-1), mononuclear phagocyte colony-stimulating factor (M-CSF),neurotrophic factors (NT3), tissue inhibitor of metalloproteinases(TIMP-1), TIMP-2, tumor necrosis factor (TNF-β), vascular endothelialgrowth factor (VEGF), VEGF-D, urokinase plasminogen activator receptor(uPAR), bone morphogenetic protein 4 (BMP4), IL1-a, IL-3, leptin, stemcell factor (SCF), stromal cell-derived factor-1 (SDF-1), plateletderived growth factor-BB (PDGFBB), transforming growth factors beta(TGFβ-1) and TGFβ-3.

In some embodiments, the composition comprising the disclosed EVsfurther comprises a protease inhibitor, RNAse inhibitor, or combinationthereof.

Also disclosed is a method of treating a subject with a with a spinalcord injury, stroke, traumatic brain injury or a neurodegenerativedisease comprising administering to the subject an effective amount of acomposition containing a neural EVs disclosed herein. In someembodiments, the neurodegenerative disease is Alzheimer's disease,Parkinson's disease, a Parkinson's-related disorder, Huntington'sdisease, prion disease, motor neuron disease (MND), spinocerebellarataxia (SCA) or spinal muscular atrophy (SMA).

There are several protein families represented in the contents of thedisclosed EV that could be beneficial in the context of several agerelated diseases. These include catalytically active enzymes likemetalloproteases, several calcium-mediated signaling proteins, and otherion channels. Several DNA and RNA polymerase subunits are present aswell as ubiquitin ligases, and proteasome subunits. Therefore, alsodisclosed is a method of treating a subject with a protein homeostasisdisorder or a proteinopathy comprising administering to the subject aneffective amount of a composition containing a neural EVs disclosedherein.

Also disclosed is the use of a composition containing a neural EVs inthe manufacture of a medicament for treating a patient or subject with aspinal cord injury, who has suffered a stroke or traumatic brain injuryor has a neurodegenerative disease.

The details of one or more embodiments of the invention are set forth inthe accompa-nying drawings and the description below. Other features,objects, and advantages of the invention will be apparent from thedescription and drawings, and from the claims.

DESCRIPTION OF DRAWINGS

FIG. 1 illustrates potential sources of cells from which extracellularvesicles (EVs) (e.g. exosomes) may be obtained.

FIG. 2 is an ultrastructural analysis indicating that multivesicularbodies (MVBs) which contain EVs are common in neural progenitors (NPs).Traditionally described as “vesicles containing smaller spherical andellipsoidal vesicles and other inclusions described as filaments,granules, irregular dense masses, and membrane components”, are apparentnear the limiting membrane of NPs. Also as reported, the vesicles werefrequently found in clusters of 3 or more vesicles (panels A, B). Theinset from panel B is shown in panel C and this MVB actual has a vesiclebudding inward into the larger multivesicular body, supporting a rolefor these vesicles in protein recycling from the plasma membrane. Thesevesicles do appear to bind with the plasma membrane, releasing EVs intothe extracellular space (panels D, E). Previous reports indicate thatthe vesicles can coalesce, and this seems common in NPs, and can be seenin most cells with clusters of MVBs (panels A, F, G). Panel H is atransmission electron microscopic (TEM) image of purified EVs from NPs.

FIGS. 3A to 3D show that EVs can be purified and detected bynanoparticle tracking analysis. FIG. 3A shows proteins from exosomesseparated by SDS-PAGE and gels stained with coomassie stain. Proteinprofiles from EVs overlapped but was distinctive from the cell pellet,supporting that cargoes are specifically trafficked to EVs. FIG. 3Bshows that protein content when compared by BCA Assay reveals thatamount of protein in the profile changes when cells are exposed tostressful conditions, like nutrient deprivation. FIGS. 3C and 3D showNanoSight analysis of purified EVs from neural progenitor cells (FIG.3C) and astrocytes (FIG. 3D). These EVs show overlapping but differentsize profiles, with both types of cells producing a peak indicating 55nm vesicles, and ranging from 25-250 nm.

FIG. 4 shows differentiated neural cells internalize DiI-labeled EVsfrom neural progenitor cells. By adding 10 uM DiI to the cellularsupernatant for 30 minutes prior to the final EVs spin and PBS wash, itwas possible to label EVs. When added into the culture medium ofdifferentiated cells, uptake of EVs was evident within 5 minutes.

FIGS. 5A and 5B show EVs from neural progenitors protect moredifferentiated neural cells from starvation stress. FIG. 5A shows EVsharvested by ultrafiltration from neural progenitor cells (NPs),astrocytes, or human umbilical MSCs. Protein content was measured byBCA, and EVs were serially diluted and transferred in equal volumesphosphate buffered serum (PBS) into wells containing NeuroNet cells (6-8week differentiated). Cells were subjected to starvation stress for 10days, when cells were fixed, and stained for β-III tubulin (Tuj). Thecenter 20 fields of view per well were imaged using the CellomicsArrayscan (representative images from 4 technical reps shown). FIG. 5Bshows NP and Astrocyte EVs protected the cells from starvations stress,and largely maintained integrity of the monolayer and extensions. Whilemore cells and debris are present in the MSC treated wells, the cellshad largely lost their neural morphology. Few cells were stilldetectable in the wells that received only PBS. NP and Astrocyte EVssamples had higher protein concentrations, so were treated with 50 μgprotein/well. Protein was limiting in the MSC samples, so 6.25 was thehighest concentration possible, but still appeared to kill the cells ina concentration dependent way, so it is unlikely that the higher doses(50, 25, or 12.5 μg/well) would produce a different result.

FIGS. 6A to 6C show indium-111 labeled EVs can be found in proximity ofstroke tissue within 1 hour of injection. FIGS. 6A to 6C showbiodistribution of indium-111 labeled EVs (FIG. 6B, 6C) compared to freeindium (FIG. 6A), which indicates that EVs are present in the brain inproximity of the stroke within 1 hour of injection either when injectedimmediately following stroke (FIG. 6B circles, left panels), or 24 hoursafter the stroke occurred (FIG. 6C, circles, left panels). Regardless ofthe timing of the initial injection, EVs were largely cleared from thearea 24 hours after injection (FIGS. 6B, 6C; circles, right panels)although a smaller amount of radioactivity was still detectable, likelyindicating EVs were metabolized by surrounding tissues.

FIG. 7 shows biodistribution of DiR labeled EVs in piglet brain. DiRlabeled EVs (approximately 2.7×10¹⁰ vesicles/kg) were detectable fromboth the dorsal and ventral via direct delivery into the brainparenchyma by stereotaxic injection (top panels), and into the CSF inthe subarachnoid space (lower panels). Fluorescence was detectable inthe dorsal and ventral aspects of the brain in both instances, but morepronounced in the dorsal region when injected IP (top panels), andventral areas of the brain following CSF injection (lower panels).

FIGS. 8A to 8E show treatment using EVs derived from neural cellsimproves infarct size and functional outcomes in the mouse embolicstroke model. APEX, MSCEX, PBS, and NPEX aliquots were provided toblinded investigators for injection into mice following induction ofembolic stroke (3 repeated doses of approximately 2.7×10¹¹ vesicles/kgat 2, 14, and 28 hours after stroke). TTC staining of brains harvested96 hours post-stroke revealed substantially decreased infarct sizefollowing NPEX or APEX treatment while MSCEX had no effect (FIG. 8A,8C). NPEX and APEX also decreased mortality over the course of the studywith over 40% of control and MSCEX treated mice succumbing tocomplications over the course of the study while approximately 25-30% ofAPEX and NPEX treated animals were lost (FIG. 8B). Phenotypic benefitsincluding improved sensory capacity detected by adhesive tape test (FIG.8E), and fewer neurological deficits as summarized by neurologicaldeficit score (FIG. 8D) were also noted in APEX and NPEX treatedanimals.

FIGS. 9A to 9C show EVs derived from neural cells modulate the immuneresponse following stroke. Blood sample flow cytometry 96 hourspost-stroke indicates that APEX and NPEX increase regulatory T-cellsrelative to MSCEX and control (vehicle) treated groups (FIG. 9A),decrease inflammatory T-helper cells (FIG. 9B), and increaseanti-inflammatory M2 macrophages in the circulation (FIG. 9C).

FIGS. 10A to 10D show NPEX treatment results in decreased infarct volumewithin 28±4 hours post-stroke as measured by MRI. Animals subjected tostroke received either NPEX (approximately 2.7×10¹⁰ vesicles/kg) or PBS(vehicle) at 2, 14, and 24 hours post-stroke. While anesthetized,animals were also subjected to MRI analysis. NPEX treated animals hadsubstantially reduced infarct volume compared to those that received PBS(FIGS. 10A-10C). When the size difference between the ipsilateral andcontralateral sides was evaluated, there was less difference betweenhemispheres in the NPEX group, indicating less edema and swelling ascompared to controls (FIG. 10D).

FIG. 11 shows NPEX treatment improves molecular deficits 12 weeks afterstroke. T2 and T2 flair images both indicate less detectable damage inNPEX treated pigs 12 weeks post-stroke. The absence (death) of tissuewas significantly reduced at the 12 week timepoint and areas of densityincongruent with host tissue were far less evident in treated animals.

FIG. 12 is a Venn diagram showing the number of proteins unique to, andshared by NPEX, APEX, and MSCEX EVs.

DETAILED DESCRIPTION Definitions

In accordance with the present invention there may be employedconventional cell culture methods, chemical synthetic methods and otherbiological and pharmaceutical techniques within the skill of the art.Such techniques are well-known and are otherwise explained fully in theliterature.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise (such as in the case of a groupcontaining a number of carbon atoms in which case each carbon atomnumber falling within the range is provided), between the upper andlower limit of that range and any other stated or intervening value inthat stated range is encompassed within the invention. The upper andlower limits of these smaller ranges may independently be included inthe smaller ranges is also encompassed within the invention, subject toany specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either bothof those included limits are also included in the invention.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It is to be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Furthermore, the following terms shall have the definitions set outbelow. It is understood that in the event a specific term is not definedherein below, that term shall have a meaning within its typical usewithin context by those of ordinary skill in the art.

The term “subject” refers to any individual who is the target ofadministration or treatment. The subject can be a vertebrate, forexample, a mammal. Thus, the subject can be a human or veterinarypatient. The term “patient” refers to a subject under the treatment of aclinician, e.g., physician.

The term “therapeutically effective” refers to the amount of thecomposition used is of sufficient quantity to ameliorate one or morecauses or symptoms of a disease or disorder. Such amelioration onlyrequires a reduction or alteration, not necessarily elimination.

The term “pharmaceutically acceptable” refers to those compounds,materials, compositions, and/or dosage forms which are, within the scopeof sound medical judgment, suitable for use in contact with the tissuesof human beings and animals without excessive toxicity, irritation,allergic response, or other problems or complications commensurate witha reasonable benefit/risk ratio.

The terms “treat”, “treating”, and “treatment”, etc., as used herein,refer to any action providing a benefit to a patient at risk for orafflicted by a disease state, condition or deficiency which may beimproved using cellular compositions according to the present invention.Treating a condition includes improving the condition through lesseningor suppression of at least one symptom, delay in progression of theeffects of the disease state or condition, including the prevention ordelay in the onset of effects of the disease state or condition, etc. Inthe present application, treatment can involve reducing the impact of aspinal cord injury or stroke, including reversing and/or inhibiting theeffects of such injury, reversing, improving, inhibiting and/orstabilizing a neurodegenerative disease such that the disease improvesand/or does not progress or worsen. The term “prophylactic” is used todescribe a method which “reduces the likelihood” that a particularresult will occur, often the progression and/or worsening of a diseasestate and/or condition.

Standard techniques for growing cells, separating cells, and whererelevant, cloning, DNA isolation, amplification and purification, forenzymatic reactions involving DNA ligase, DNA polymerase, restrictionendonucleases and the like, and various separation techniques are thoseknown and commonly employed by those skilled in the art. A number ofstandard techniques are described in Sambrook et al., 1989 MolecularCloning, Second Edition, Cold Spring Harbor Laboratory, Plainview, N.Y.;Maniatis et al., 1982 Molecular Cloning, Cold Spring Harbor Laboratory,Plainview, N.Y.; Wu (Ed.) 1993 Meth. Enzymol. 218, Part I; Wu (Ed.) 1979Meth. Enzymol. 68; Wu et al., (Eds.) 1983 Meth. Enzymol. 100 and 101;Grossman and Moldave (Eds.) 1980 Meth. Enzymol. 65; Miller (Ed.) 1972Experiments in Molecular Genetics, Cold Spring Harbor Laboratory, ColdSpring Harbor, N.Y.; Old and Primrose, 1981 Principles of GeneManipulation, University of California Press, Berkeley; Schleif andWensink, 1982 Practical Methods in Molecular Biology; Glover (Ed.) 1985DNA Cloning Vol. I and II, IRL Press, Oxford, UK; Hames and Higgins(Eds.) 1985 Nucleic Acid Hybridization, IRL Press, Oxford, UK; andSetlow and Hollaender 1979 Genetic Engineering: Principles and Methods,Vols. 1-4, Plenum Press, New York. Abbreviations and nomenclature, whereemployed, are deemed standard in the field and commonly used inprofessional journals such as those cited herein.

The term “human Pluripotent Stem Cells”, of which “human Embryonic StemCells” (hESCs) and human induced pluripotent stem cells (hiPSCs) are asubset, are derived from pre-embryonic, embryonic, fetal tissue or adultstem cells (in the case of human induced pluripotent stem cells) at anytime after fertilization, and have the characteristic of being capableunder appropriate conditions of producing progeny of several differentcell types, especially including neuronal stem and progenitors, neuralcrest cells, mesenchymal stem cells (MSCs) and related proliferative andnon-proliferative neural cells. The term includes both established linesof stem cells of various kinds, and cells obtained from primary tissuethat are pluripotent in the manner described.

The term “embryonic stem cell” refers to pluripotent cells, preferablyof primates, including humans, which are isolated from the blastocyststage embryo.

The term “neural progenitor cell” refers to cells capable of dividing alimited number of times that have the capacity to differentiate intoneuronal and glial cell types.

The terms “extracellular vesicle” and “EV” are used herein to refer to avesicle of about 10 nm to 10 μm in size consisting of fluid,macro-molecules, solutes, and metabolites from a cell contained by alipid bilayer or micelle. In some cases, the EV is a cell-derived EV.The term “EV” also includes lipid vesicle engineered to containbioactive molecules found in a cell-derived EVs, such as a neural EVs.These terms encompass both exosomes and ectosomes. Exosomes are releasedon the exocytosis of multivesicular bodies (MVBs). Ectosomes arevesicles assembled at and released from the plasma membrane. In somecases, the EV is about 20 nm to 10 μm, 20 nm to 1 μm, 20 nm-500 nm, 30nm-100 nm, 30 nm-160 nm, or 80-160 nm in size. In some embodiments, theEVs are exosomes that are about 20 to 150 nm in size.

The term “autologous EV” is used to describe a population of EVs whichare obtained from cells from a subject or patient to whom the EVs are tobe administered.

The term “neural EV” is used to refer to a cell-derived EV produced fromneural progenitor cells derived in vitro from pluripotent stem cells orneural cells derived in vitro from said neural progenitor cells or frompluripotent stem cells. The term also refers to vesicles engineered tocontain a sufficient number of the bioactive molecules found in thecell-derived neural EV to have substantially the same bioactivity.

Compositions

Disclosed herein are neural EVs (e.g. exosomes) and methods of usingthese EVs in the treatment of spinal cord injury, stroke, and traumaticbrain injury and neurodegenerative diseases.

The disclosed EVs can be obtained in some embodiments, by culturingneural progenitor (NP) cells or mesenchymal stem cells (MSCs) that wereproduced in vitro from pluripotent stem cells (e.g. human embryonic stemcells (hESCs) or induced pluripotent stem cells (iPSCs)) in cell culturemedium under conditions and for a time sufficient for the NP cells orMSCs to produce EVs.

Methods for the production of human neural progenitor (hNP) cells fromhuman embryonic stem cells (ESCs) are described, for example, in U.S.Pat. No. 7,531,354, which is hereby incorporated by reference in itsentirety for the teaching of these cells. Human neuroprogenitor cells(hNPs) are known to express markers associated with the earliestmultipotent neural stem cells, including Nestin, Musashi-1, Sox1, Sox2and Sox3. It is noted that although feeder cell free neural progenitorcells may be used to produce EVs, any neuroprogenitor cell as otherwisedescribed herein may also be used. Preferred neuroprogenitor cells areproduced according to the methods presented in U.S. Pat. No. 7,531,354,are adherent feeder cell free as well as free from embryoid bodies.

The disclosed EVs can be obtained in some embodiments by culturingdifferentiated neural cells, such as astrocytes, derived directly orindirectly from pluripotent stem cells in cell culture medium underconditions and for a time sufficient to produce EVs, and isolating saidEVs from the culture medium. In some embodiments, the differentiatedneural cells are hN2™ neuronal cells (ArunA Biomedical), NeuroNet™neurons, or AstroPro™ astrocytes (ArunA Biomedical Inc).

Pluripotent stem cells used to produce the EV-producing NP cells, neuralcells, or MSCs include human embryonic stem cells (hESCs) and humaninduced pluripotent stem cells (hiPSCs).

Pluripotent stem cells may express one or more of the stage-specificembryonic antigens (SSEA) 3 and 4, and markers detectable usingantibodies designated Tra-1-60 and Tra-1-81 (Thomson et al., Science282:1145, 1998). Differentiation of pluripotent stem cells in vitroresults in the loss of SSEA-4, Tra-1-60, and Tra-1-81 expression (ifpresent) and increased expression of SSEA-1. Undifferentiatedpluripotent stem cells typically have alkaline phosphatase activity,which can be detected by fixing the cells with 4% paraformaldehyde, andthen developing with Vector Red as a substrate, as described by themanufacturer (Vector Laboratories, Burlingame Calif.) Undifferentiatedpluripotent stem cells also typically express Oct-4 and TERT, asdetected by RT-PCR.

The types of pluripotent stem cells that may be used include establishedlines of pluripotent cells derived from tissue formed after gestation,including pre-embryonic tissue (such as, for example, a blastocyst),embryonic tissue, or fetal tissue taken any time during gestation,typically but not necessarily before approximately 10-12 weeksgestation. Non-limiting examples are established ethical lines of humanembryonic stem cells or human embryonic germ cells, such as, for examplethe human embryonic stem cell lines WA01, WA07, and WA099 (WiCell). Alsocontemplated is use of the compositions of this disclosure during theinitial establishment or stabilization of such cells, in which case thesource cells would be primary pluripotent cells taken directly from thesource tissues. Also suitable are cells taken from a pluripotent stemcell population already cultured in the absence of feeder cells. Alsosuitable are mutant human embryonic stem cell lines, such as, forexample, BG01v (BresaGen, Athens, Ga.), as well as normal humanembryonic stem cell lines such as WA01, WA07, WA09 (WiCell) and BG01,BG02 (BresaGen, Athens, Ga.).

Human embryonic stem cells (hESCs) may be prepared by methods which aredescribed in the in the art as described for example, by Thomson et al.(U.S. Pat. No. 5,843,780; Science 282:1145, 1998; Curr. Top. Dev. Biol.38:133 ff., 1998; Proc. Natl. Acad. Sci. U.S.A. 92:7844, 1995).Alternatively, they may be obtained commercially.

Epiblast stem cells (EpiScs) and induced pluripotent stem cells (iPSCs)isolated from early post-implantation stage embryos. They express Oct4and are pluripotent. iPSCs are made by dedifferentiating adult somaticcells back to a pluripotent state by retroviral transduction of fourgenes (c-myc, Klf4, Sox2, Oct4).

As described in U.S. Patent Application Document No. 20140356382,“[e]xosomes produced from cells can be collected from the culture mediumand/or cell tissue by any suitable method. Typically a preparation ofEVs can be prepared from cell culture or tissue supernatant bycentrifugation, filtration or combinations of these methods. Forexample, EVs can be prepared by differential centrifugation, that is lowspeed (<2,0000 g) centrifugation to pellet larger particles followed byhigh speed (>100,000 g) centrifugation to pellet EVs, size filtrationwith appropriate filters (for example, 0.22 μm filter), gradientultracentrifugation (for example, with sucrose gradient) or acombination of these methods.” It is noted that the contents of EVs,i.e., EVs in which the lipid bilayer has been removed or eliminated andthe contents obtained may also be used to engineer artificial EVs.

Further, as described in U.S. Patent Application Document No.20140356382, exogenous protein and/or peptide and other cargo can beintroduced into the EVs by a number of different techniques includingelectroporation or the use of a transfection reagent. Electroporationconditions may vary depending on the charge and size of thebiotherapeutic cargo. Typical voltages are in the range of 20V/cm to1,000V/cm, such as 20V/cm to 100V/cm with capacitance typically between25 μF and 250 μF, such as between 25 μF and 125 μF. A voltage in therange of 150 mV to 250 mV, particularly a voltage of 200 mV is preferredfor loading EVs with an antibody. Alternatively, the EVs may be loadedwith exogenous protein and/or peptide using a transfection reagent.Despite the small size of the EVs, conventional transfection agents maybe used for transfection of EVs with protein and/or peptide. EVs mayalso be loaded by transforming or transfecting a host cell with anucleic acid construct which expresses therapeutic protein or peptide ofinterest, such that the therapeutic protein or peptide is taken up intothe EVs as the EVs are produced from the cell.

In illustrative embodiments, the EV-producing NP cells and/or neuralcells disclosed herein are cultured for about 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13 or 14 days or for as long as about 1, 2, 3, 4, 5, 6, 7, 8weeks or about 1, 2, 3, 4, 5, or 6 months, depending on the cell and itsability to produce EVs. The EV-producing cells may be cultured insuitable media and grown under conditions that are readily determined byone of ordinary skill in the art. Cell culture conditions may vary withcell type and the examples presented hereinafter illustrate suitablemedia and conditions. For example, CMRL 1066 medium (from Invitrogen)with fetal bovine serum (e.g., at 10%) and optionally supplemented withglutamine or glutamine-containing mixtures and antibiotics could beused. Cells can be grown on a surface (feeder cells) in someembodiments, e.g. they can be grown as a monolayer on the surface(feeder cell free) and may be grown until 30, 40, 50, 60, 70, 80, 90, 95or 100% confluent.

Cell growth media are well known in the art and comprise at least aminimum essential medium plus one or more optional components such asgrowth factors, ascorbic acid, glucose, non-essential amino acids, salts(including trace elements), glutamine, insulin (where indicated and notexcluded), Activin A, transferrin, beta mercaptoethanol, and otheragents well known in the art and as otherwise described herein. Apreferred media is a low protein, serum-free based growth medium thatsupports neural cells. The growth factor used can be fibroblast growthfactor 2 (FGF2), alone or preferably in combination with leukemiainhibitor factor (LIF). Depending on the NP or neural cells to be grownin the growth media, the inclusion of LIF is preferred but may not berequired. Additional media includes basal cell media which may containserum, for example, between about 0.1% and 20% (preferably, about 2-10%)fetal calf serum, or for defined medium, an absence of fetal calf serumand KSR, and optionally including bovine serum albumin (about 1-5%,preferably about 2%). Preferred medium is defined and is serum-free andlow protein. The components of the growth media depends on the type ofneural cell to be grown, all of which are well known in the art.Particularly preferred media is media and supplement from ArunA. TheAB2™ Neural Cell Culture Media Kit contains AB2™ Basal Neural Medium andANS™ Neural Medium Supplement. The medium and supplement arespecifically engineered for versatility to meet all neural cell cultureneeds. The AB2™ Basal Neural Medium and ANS™ Neural Medium Supplementcan be used as the base for specialized mediums to directdifferentiation of the hNP1™ line toward various neural phenotypes. Eachlot of medium and supplement is pre-qualified for use by testing forcell growth, sterility, pH, osmolarity, and endotoxins.

Formulations from ArunA allow neural cultures to maintain a stablekaryotype over multiple passages without the need for feeder cells,making them an excellent choice for a wide variety of researchapplications including early stage drug discovery.

Other agents which optionally may be added to the medium include,depending on the cell type grown in the media, for example, any one ormore of nicotinamide, members of TGF-β family, including TGF-β 1, 2, and3, Activin A, nodal, Bone Morphogen Proteins (BMP 2 to 7) serum albumin,members of the fibroblast growth factor (FGF) family, platelet-derivedgrowth factor-AA, and —BB, platelet rich plasma, insulin growth factor(IGF-I, II, LR-IGF), growth differentiation factor (GDF-5, -6, -8, -10,11), glucagon like peptide-I and II (GLP-I and II), GLP-1 and GLP-2mimetobody, Exendin-4, parathyroid hormone, insulin, progesterone,aprotinin, hydrocortisone, ethanolamine, epidermal growth factor (EGF),gastrin I and II, copper chelators such as, for example, triethylenepentamine, forskolin, Na-Butyrate, betacellulin, ITS, noggin, neuritegrowth factor, nodal, valporic acid, trichostatin A, sodium butyrate,hepatocyte growth factor (HGF), sphingosine-1, VEGF, MG132 (EMD, CA), N2and B27 supplements (Gibco, CA), steroid alkaloid such as, for example,cyclopamine (EMD, CA), keratinocyte growth factor (KGF), Dickkopfprotein family, bovine pituitary extract, islet neogenesis-associatedprotein (INGAP), Indian hedgehog, sonic hedgehog, proteasome inhibitors,notch pathway inhibitors, sonic hedgehog inhibitors, heregulin, orcombinations thereof, among a number of other components. Each of thesecomponents, when included, are included in effective amounts.

By way of further example, suitable media may be made from the followingcomponents, such as, for example, Dulbecco's modified Eagle's medium(DMEM), Gibco #11965-092; Knockout Dulbecco's modified Eagle's medium(KO DMEM), Gibco #10829-018; Ham's F12/50% DMEM basal medium; 200 mML-glutamine, Gibco #15039-027; non-essential amino acid solution, Gibco11140-050; β-mercaptoethanol, Sigma #M7522; human recombinant basicfibroblast growth factor (bFGF), Gibco #13256-029.

Cell media are commercially available and can be supplemented withcommercially available components, including defined xeno-freecomponents, such as those available from Invitrogen Corp. (GIBCO), CellApplications, Inc., Biological Industries, Beth HaEmek, Israel, andCalbiochem. One of ordinary skill in the art will be able to readilymodify the cell media to produce any one or more of the target cellspursuant to the present invention.

The disclosed EV-producing cells may be cultured on a layer of feedercells that support the cells in various ways. Approaches for culturingcells on a layer of feeder cells are well known in the art. The cellsmay be grown on a cellular support or matrix, as adherent monolayers,rather than as embryoid bodies or in suspension. In certain embodiments,the use of a cellular support may be preferred, depending upon the cellsused to produce the EVs. When used, cellular supports preferablycomprise at least one substrate protein. Substrate proteins include, forexample, an extracellular matrix protein, which is a protein found inthe extracellular matrix, such as laminin, tenascin, thrombospondin, andmixtures thereof, which exhibit growth promoting and contain domainswith homology to epidermal growth factor (EGF) and exhibit growthpromoting activity. Other substrate proteins which may be used includefor example, collagen, fibronectin, vibronectin, polylysine,polyornithine and mixtures thereof. In addition, gels and othermaterials such as methylcellulose of other gels which contain effectiveconcentrations of one or more of these embryonic stem celldifferentiation proteins may also be used. Exemplary differentiationproteins or materials which include these differentiation proteinsinclude, for example, laminin, BD Cell-Tak™ Cell and Tissue Adhesive,BD™ FIBROGEN Human Recombinant Collagen I, BD™ FIBROGEN HumanRecombinant Collagen III, BD Matrigel™ Basement Membrane Matrix, BDMatrigel™ Basement Membrane Matrix High Concentration (HC), BD™PuraMatrix™ Peptide Hydrogel, Collagen I, Collagen I High Concentration(HC), Collagen II (Bovine), Collagen III, Collagen IV, Collagen V, andCollagen VI, among others.

Alternatively, these cells may be cultured in a culture system that isessentially free of feeder cells, but nonetheless supports proliferationof the cells to produce EVs. The growth of cells in feeder-free culturecan be supported using a medium conditioned by culturing previously withanother cell type. Alternatively, the growth of EV-producing cells infeeder-free culture without differentiation can be supported using achemically defined medium. These approaches are well known in the art.In certain embodiments of the present invention, the cells are grown infeeder cell free medium.

EVs can be harvested at various time intervals (e.g. at about 2, 4, 6, 8or 3, 6, 9, 12 day or longer intervals, depending upon the rate ofproduction of EVs). Exemplary yields of EVs can range from at leastabout 1 ng EVs/1 million cells, at least about 10 ng EVs/1 millioncells, at least about 50 ng EVs/1 million cells, at least about 100 ngEVs/1 million cells, at least about 500 ng EVs/1 million cells, at leastabout 750 ng EVs/1 million cells, at least about 800 ng EVs/1 millioncells, at least about 900 ng EVs/1 million cells, at least about 1.0 μgEVs/1 million cells, at least about 1.5 μg EVs/1 million cells, at leastabout 2.0 μg EVs/1 million cells, at least about 2.5 μg EVs/1 millioncells, at least e.g. about 3.0 μg EVs/1 million cells, at least about5.0 μg EVs/1 million cells, and at least about 10.0 μg EVs/1 millioncells, during a time period of about 24 hours to seven days of cultureof proliferative and non-proliferative neural cells as otherwisedescribed herein.

In certain embodiments, EVs are harvested and collected byultracentrifugation or differential centrifugation or any combinationthereof, pelleted EVs are collected, and, optionally, collected pelletedEVs are washed with a suitable medium. For example, a preparation of EVscan be prepared from cell culture or tissue supernatant bycentrifugation, filtration or combinations of these methods. In someembodiments, the EVs can be prepared by differential centrifugation,that is low speed (<2,0000 g) centrifugation to pellet larger particlesfollowed by high speed (>100,000 g) centrifugation to pellet EVs, sizefiltration with appropriate filters (for example, 0.22 μm filter),gradient ultracentrifugation (for example, with sucrose gradient) or acombination of these methods. EVs may be purified by differentialcentrifugation, micro and ultra-filtration, polymeric precipitation,microfluidic separation, immunocapture and size-exclusionchromatography. These and/or related methods for isolating and purifyingEVs are described by Théry, et al., Current Protocols in Cell Biology,(2006) 3.221-3.22.29, copyright 2006 by John Wiley & Sons, Inc.;Sokolova, et al., Colloids and Surfaces B: Biointerfaces, 2011, 87,146-150; Wiklander, et al., Journal of Extracellular Vesicles, 2015, 4,26316, pp. 1-13; and Böing, et al., Journal of Extracellular Vesicles,2014, 3, 23430, pp. 1-11. Other methods for isolation may be developedsuch as electrical field radiofrequency and acoustics.

Pharmaceutical Compositions

Disclosed is a pharmaceutical compositions containing therapeuticallyeffective amounts of one or more of the disclosed EVs and apharmaceutically acceptable carrier. Formulations containing thedisclosed EVs may take the form of liquid, solid, semi-solid orlyophilized powder forms, such as, for example, solutions, suspensions,emulsions, sustained-release formulations, tablets, capsules, powders,suppositories, creams, ointments, lotions, aerosols, patches or thelike, preferably in unit dosage forms suitable for simple administrationof precise dosages.

Pharmaceutical compositions typically include a conventionalpharmaceutical carrier and/or excipient and may additionally includeother medicinal agents, carriers, adjuvants, additives and the like. Theweight percentage ratio of the EVs to the one or more excipients can bebetween about 20:1 to about 1:60, or between about 15:1 to about 1:45,or between about 10:1 to about 1:40, or between about 9:1, 8:1, 7:1,6:1, 5:1, 4:1, 3:1, 2:1 or 1:1 to about 1:2, 1:3, 1:4, 1:5, 1:6, 1:7,1:8, 1:9, 1:10, 1:15, 1:20, 1:25, 1:30, or 1:35, and preferably is about20:1, 19:1, 18:1, 17:1, 16:1, 15:1, 14:1, 13:1, 12:1, 11:1, 10:1, 9:1,8:1, 7:1, 6:1 or 5:1. In some embodiments, the disclosed compositioncomprises between about 1 μg to about 1 g or more of total EVs, about500 μg about 500 mg, about 1 mg to about 500 mg of total EVs, about 5 toabout 500 mg, about 10 to about 500 mg, about 25 to about 500 mg, about50 mg to about 350 mg, about 75 mg to about 450 mg, about 50 mg to about450 mg, or about 75 mg to about 325 mg or about 100 mg to about 650 mgof total EVs and may optionally contain one or more suitablepharmaceutical carriers, additives and/or excipients.

An injectable composition for parenteral administration (e.g.intravenous, intramuscular, intrathecal intracerebrospinal fluid, orintranasal), will typically contain the EVs and optionally additionalcomponents in a suitable i.v. solution, such as sterile physiologicalsalt solution. The composition may also be formulated as a suspension inan aqueous emulsion.

Liquid compositions can be prepared by dissolving or dispersing thepharmaceutical composition comprising the EVs, and optionalpharmaceutical adjuvants, in a carrier, such as, for example, aqueoussaline, aqueous dextrose, glycerol, or ethanol, to form a solution orsuspension. For use in an oral liquid preparation, the composition maybe prepared as a solution, suspension, emulsion, or syrup, beingsupplied either in liquid form or a dried form suitable for hydration inwater or normal saline. In the case of intranasal, intratracheal orintrapulmonary administration, the compositions may be provided asliquid composition which can be sprayed into the nose, trachea and/orlungs.

For oral administration, such excipients include pharmaceutical gradesof mannitol, lactose, starch, magnesium stearate, sodium saccharine,talcum, cellulose, glucose, gelatin, sucrose, magnesium carbonate, andthe like. If desired, the composition may also contain minor amounts ofnon-toxic auxiliary substances such as wetting agents, emulsifyingagents, or buffers.

When the composition is employed in the form of solid preparations fororal administration, the preparations may be tablets, granules, powders,capsules or the like. In a tablet formulation, the composition istypically formulated with additives, e.g. an excipient such as asaccharide or cellulose preparation, a binder such as starch paste ormethyl cellulose, a filler, a disintegrator, and other additivestypically used in the manufacture of medical preparations.

Methods for preparing such dosage forms are known or are apparent tothose skilled in the art; for example, see Remington's PharmaceuticalSciences (17th Ed., Mack Pub. Co. 1985). The composition to beadministered will contain a quantity of the selected compound in apharmaceutically effective amount for therapeutic use in a biologicalsystem, including a patient or subject according to the presentinvention.

Intravenous formulations can comprise the EVs described herein, anisotonic medium and one or more substances preventing aggregation of theEVs. Example intravenous/intrathecal/intracerebrospinal fluidformulations may contain saline solutions (e.g. normal saline (NS);about 0.91% w/v of NaCl, about 300 mOsm/L) and/or dextrose 4% in 0.18%saline, and optionally 1%, 2% or 3% human serum albumin. In addition,the EVs may be disrupted to obtain the contents and the contents used incompositions according to the present invention.

In exemplary embodiments, formulations of the invention may compriseabout 50 ng EVs/ml intravenous/intrathecal/intracerebrospinal fluidmedium, including about 100 ng, 200 ng, 300 ng, 400 ng, 500 ng, 600 ng,700 ng, 800 ng, 900 ng, 1.0 μg, 1.5 μg, 2.0 μg, 2.5 μg, 3.0 μg, 5.0 μg,10.0, 15.0 μg, 20.0 μg, 100 μg, or more EVs/mlintravenous/intrathecal/intracerebrospinal fluid medium for use intreating spinal cord injury, stroke, traumatic brain injury and/orneurodegenerative diseases.

In some embodiments, intravenous formulations may comprise about 0.1 μgEVs/ml medium, about 0.2 μg EVs/ml intravenous medium, about 0.3 μgEVs/ml intravenous medium, about 0.4 μg EVs/ml intravenous medium, about0.5 μg EVs/ml intravenous medium, about 0.6 μg EVs/ml intravenousmedium, about 0.7 μg EVs/ml intravenous medium, about 0.8 μg EVs/mlintravenous medium, about 0.9 μg EVs/ml intravenous medium, about 1.0 μgEVs/ml intravenous medium, about 1.5 μg EVs/ml intravenous medium, about2.0 μg EVs/ml intravenous medium, about 2.5 μg EVs/ml intravenousmedium, such as at least e.g. about 3.0 μg EVs/ml intravenous medium,such as e.g. at least about 5.0 μg EVs/ml intravenous medium, about 10.0μg EVs/ml intravenous medium, 15.0 μg EVs/ml intravenous medium or about20.0 μg or more EVs/ml intravenous medium.

In some embodiments, the pharmaceutical composition is in a dosage formcomprising at least 25 mg of EVs, at least 50 mg of EVs, at least 60 mgof EVs, at least 75 mg of EVs, at least 100 mg of EVs, at least 150 mgof EVs, at least 200 mg of EVs, at least 250 mg of EVs, at least 300 mgof EVs, about 350 mg of EVs, about 400 mg of EVs, about 500 mg of EVs,about 750 mg of EVs, about 1 g (1,000 mg) or more of EVs, alone or incombination with a therapeutically effective amount of at least oneadditional bioactive agent, which agent may be useful in the treatmentof spinal cord injury, stroke, traumatic brain injury and/orneurodegenerative disease. In some embodiments, the pharmaceuticalcomposition comprises between about 10 mg to about 750 mg, about 25 mgto about 650 mg, or between about 30 mg to about 500 mg, or about 35 mgto about 450 mg, most often about 50 to about 500 mg of EVs.

In some embodiments, an intravenous formulation comprises the EVsdescribed herein, an isotonic medium, and one or more substancespreventing aggregation of the EVs. Intravenous formulations maytherefore contain saline solutions (e.g. normal saline (NS); about 0.91%w/v of NaCl, about 300 mOsm/L) and/or dextrose 4% in 0.18% saline, andoptionally 1%, 2% or 3% human serum albumin.

In some embodiments, the composition comprising the disclosed EVsfurther comprises one more neurotrophic agents. The composition canfurther comprises one or more agents selected from the group consistingof leukemia inhibitory factor (LIF), brain-derived neurotrophic factor(BDNF), epidermal growth factor receptor (EGF), basic fibroblast growthfactor (bFGF), FGF-6, glial-derived neurotrophic factor (GDNF),granulocyte colony-stimulating factor (GCSF), hepatocyte growth factor(HGF), IFN-γ, insulin-like growth factor binding protein (IGFBP-2),IGFBP-6, IL-1ra, IL-6, IL-8, monocyte chemotactic protein (MCP-1),mononuclear phagocyte colony-stimulating factor (M-CSF), neurotrophicfactors (NT3), tissue inhibitor of metalloproteinases (TIMP-1), TIMP-2,tumor necrosis factor (TNF-β), vascular endothelial growth factor(VEGF), VEGF-D, urokinase plasminogen activator receptor (uPAR), bonemorphogenetic protein 4 (BMP4), IL1-a, IL-3, leptin, stem cell factor(SCF), stromal cell-derived factor-1 (SDF-1), platelet derived growthfactor-BB (PDGFBB), transforming growth factors beta (TGFβ-1) andTGFβ-3.

In some embodiments, the disclosed EVs are contained in or on abiocompatible scaffold, such as a hydrogel. Suitable hydrogels includetemperature dependent hydrogels that solidify or set at bodytemperature, e.g., PLURONICS™; hydrogels crosslinked by ions, e.g.,sodium alginate; hydrogels set by exposure to either visible orultraviolet light, e.g., polyethylene glycol polylactic acid copolymerswith acrylate end groups; and hydrogels that are set or solidified upona change in pH, e.g., TETRONICS™. Examples of materials that can be usedto form these different hydrogels include polysaccharides such asalginate, polyphosphazenes, and polyacrylates, which are cross-linkedionically, or block copolymers such as PLURONICS™ (also known asPOLOXAMERS™), which are poly(oxyethylene)-poly(oxypropylene) blockpolymers solidified by changes in temperature, or TETRONICS™ (also knownas POLOXAMINES™), which are poly(oxyethylene)-poly(oxypropylene) blockpolymers of ethylene diamine solidified by changes in pH.

Suitable hydrogels also include undefined extracellular matrix derivedhydrogels that originated from tissues including but not limited tobladder intestine, blood and brain.

In some embodiments, the disclosed EVs are contained in or on abiocompatible scaffold comprising collagen, fibrin, silk, agarose,alginate, hyaluronan, chitosan, a biodegradable polyester such aspolylactic-co-glycolic acid, polylacic acid, or polyglycolic acid,polyethylene glycol, polyvinylpyrrolidone, polyethersulfone, apeptide-based biomaterial, glycose amino glycan, fibronectin, laminin,or any combination thereof.

In some cases, the hydrogel is produced by cross-linking the anionicsalt of alginic acid, a carbohydrate polymer isolated from seaweed, withions, such as calcium cations. The strength of the hydrogel increaseswith either increasing concentrations of calcium ions or alginate. Forexample, U.S. Pat. No. 4,352,883 describes the ionic cross-linking ofalginate with divalent cations, in water, at room temperature, to form ahydrogel matrix.

EVs are mixed with an alginate solution, the solution is delivered to analready implanted support structure and then solidifies in a short timedue to the presence in vivo of physiological concentrations of calciumions. Alternatively, the solution is delivered to the support structureprior to implantation and solidified in an external solution containingcalcium ions.

In general, these polymers are at least partially soluble in aqueoussolutions, e.g., water, or aqueous alcohol solutions that have chargedside groups, or a monovalent ionic salt thereof. There are many examplesof polymers with acidic side groups that can be reacted with cations,e.g., poly(phosphazenes), poly(acrylic acids), and poly(methacrylicacids). Examples of acidic groups include carboxylic acid groups,sulfonic acid groups, and halogenated (preferably fluorinated) alcoholgroups. Examples of polymers with basic side groups that can react withanions are poly(vinyl amines), poly(vinyl pyridine), and poly(vinylimidazole).

Polyphosphazenes are polymers with backbones consisting of nitrogen andphosphorous atoms separated by alternating single and double bonds. Eachphosphorous atom is covalently bonded to two side chains.Polyphosphazenes that can be used have a majority of side chains thatare acidic and capable of forming salt bridges with di- or trivalentcations. Examples of acidic side chains are carboxylic acid groups andsulfonic acid groups.

Bioerodible polyphosphazenes have at least two differing types of sidechains, acidic side groups capable of forming salt bridges withmultivalent cations, and side groups that hydrolyze under in vivoconditions, e.g., imidazole groups, amino acid esters, glycerol, andglucosyl. Bioerodible or biodegradable polymers, i.e., polymers thatdissolve or degrade within a period that is acceptable in the desiredapplication (usually in vivo therapy), will degrade in less than aboutfive years and most preferably in less than about one year, once exposedto a physiological solution of pH 6-8 having a temperature of betweenabout 25° C. and 38° C. Hydrolysis of the side chain results in erosionof the polymer. Examples of hydrolyzing side chains are unsubstitutedand substituted imidizoles and amino acid esters in which the side chainis bonded to the phosphorous atom through an amino linkage.

Methods for synthesis and the analysis of various types ofpolyphosphazenes are described in U.S. Pat. Nos. 4,440,921, 4,495,174,and 4,880,622. Methods for the synthesis of the other polymers describedabove are known to those skilled in the art. See, for example ConciseEncyclopedia of Polymer Science and Engineering, J. I. Kroschwitz,editor (John Wiley and Sons, New York, N.Y., 1990). Many polymers, suchas poly(acrylic acid), alginates, and PLURONICS™, are commerciallyavailable.

Water soluble polymers with charged side groups are cross-linked byreacting the polymer with an aqueous solution containing multivalentions of the opposite charge, either multivalent cations if the polymerhas acidic side groups, or multivalent anions if the polymer has basicside groups. Cations for cross-linking the polymers with acidic sidegroups to form a hydrogel include divalent and trivalent cations such ascopper, calcium, aluminum, magnesium, and strontium. Aqueous solutionsof the salts of these cations are added to the polymers to form soft,highly swollen hydrogels.

Anions for cross-linking the polymers to form a hydrogel includedivalent and trivalent anions such as low molecular weight dicarboxylateions, terepthalate ions, sulfate ions, and carbonate ions. Aqueoussolutions of the salts of these anions are added to the polymers to formsoft, highly swollen hydrogels, as described with respect to cations.

For purposes of preventing the passage of antibodies into the hydrogel,but allowing the entry of nutrients, a useful polymer size in thehydrogel is in the range of between 10,000 D and 18,500 D.

Temperature-dependent, or thermosensitive, hydrogels have so-called“reverse gelation” properties, i.e., they are liquids at or below roomtemperature, and gel when warmed to higher temperatures, e.g., bodytemperature. Thus, these hydrogels can be easily applied at or belowroom temperature as a liquid and automatically form a semi-solid gelwhen warmed to body temperature. As a result, these gels are especiallyuseful when the support structure is first implanted into a patient, andthen filled with the hydrogel-EV composition. Examples of suchtemperature-dependent hydrogels are PLURONICS™ (BASF-Wyandotte), such aspolyoxyethylene-polyoxypropylene F-108, F-68, and F-127,poly(N-isopropylacrylamide), and N-isopropylacrylamide copolymers.

These copolymers can be manipulated by standard techniques to affecttheir physical properties such as porosity, rate of degradation,transition temperature, and degree of rigidity. For example, theaddition of low molecular weight saccharides in the presence and absenceof salts affects the lower critical solution temperature (LCST) oftypical thermosensitive polymers. In addition, when these gels areprepared at concentrations ranging between 5 and 25% (W/V) by dispersionat 4° C., the viscosity and the gel-sol transition temperature areaffected, the gel-sol transition temperature being inversely related tothe concentration.

U.S. Pat. No. 4,188,373 describes using PLURONIC™ polyols in aqueouscompositions to provide thermal gelling aqueous systems. U.S. Pat. Nos.4,474,751, '752, '753, and 4,478,822 describe drug delivery systemswhich utilize thermosetting polyoxyalkylene gels; with these systems,both the gel transition temperature and/or the rigidity of the gel canbe modified by adjustment of the pH and/or the ionic strength, as wellas by the concentration of the polymer.

pH-dependent hydrogels are liquids at, below, or above specific pHvalues, and gel when exposed to specific pHs, e.g., 7.35 to 7.45, thenormal pH range of extracellular fluids within the human body. Thus,these hydrogels can be easily delivered to an implanted supportstructure as a liquid and automatically form a semi-solid gel whenexposed to body pH. Examples of such pH-dependent hydrogels areTETRONICS™ (BASF-Wyandotte) polyoxyethylene-polyoxypropylene polymers ofethylene diamine, poly(diethyl aminoethyl methacrylate-g-ethyleneglycol), and poly(2-hydroxymethyl methacrylate). These copolymers can bemanipulated by standard techniques to affect their physical properties.

Hydrogels that are solidified by either visible or ultraviolet light canbe made of macromers including a water soluble region, a biodegradableregion, and at least two polymerizable regions as described in U.S. Pat.No. 5,410,016. For example, the hydrogel can begin with a biodegradable,polymerizable macromer including a core, an extension on each end of thecore, and an end cap on each extension. The core is a hydrophilicpolymer, the extensions are biodegradable polymers, and the end caps areoligomers capable of cross-linking the macromers upon exposure tovisible or ultraviolet light, e.g., long wavelength ultraviolet light.

Examples of such light solidified hydrogels include polyethylene oxideblock copolymers, polyethylene glycol polylactic acid copolymers withacrylate end groups, and 10K polyethylene glycol-glycolide copolymercapped by an acrylate at both ends. As with the PLURONIC™ hydrogels, thecopolymers comprising these hydrogels can be manipulated by standardtechniques to modify their physical properties such as rate ofdegradation, differences in crystallinity, and degree of rigidity.

Methods of Treatment

Also disclosed is a method of treating a subject with a with a spinalcord injury, stroke, traumatic brain injury or a neurodegenerativedisease comprising administering to the subject an effective amount of acomposition containing a population of neural EVs disclosed herein. Insome embodiments, the neurodegenerative disease is Alzheimer's disease,Parkinson's disease, a Parkinson's-related disorder, Huntington'sdisease, prion disease, motor neuron disease (MND), spinocerebellarataxia (SCA) or spinal muscular atrophy (SMA).

The term “spinal cord injury” is used to describe a spinal cord injurywhich results in a temporary or permanent change in the normal motor,autonomic or sensory function of the cord. The damage often results fromphysical trauma, such as sports injuries, slip and fall accidents ormotor vehicular accidents but can also result from diseases such asspina bifida, Friedrich's ataxis and/or transverse myelitis. Injury tothe spinal cord resulting in a loss of function does not have to be theresult of complete severing of the spinal cord. Depending on where thespinal cord and its nerve roots are damaged, the symptoms and degree ofinjury can vary widely, from pain to incontinence to paralysis. Spinalcord injuries are described at various levels of incomplete to completeinjury, resulting in a total loss of function. The spinal cord injurycan result in paraplegia or tetraplegia.

Traditional treatment of spinal cord injuries starts with stabilizingthe spine and controlling inflammation associated with the spin corddamage to prevent further damage. Other interventions can vary widelydepending on the location and extent of the injury. In many cases, usingconventional therapy, spinal cord injuries require substantial,long-term physical therapy and rehabilitation, especially if the injuryinterferes with activities of daily life.

Spinal cord injury can be classified into three types based on itscause: mechanical forces, toxic, and ischemic, from lack of blood flow.Spinal cord damage can also be divided into primary and second injury.Primary injury is caused by the cell death that occurs immediately inthe original injury (physical trauma, exposure to toxins, or ischemia),and secondary injury is caused by the resultant cascades that are causedby the original insult and cause further tissue damage. These secondaryinjury pathways include inflammation, swelling, neurotransmitterdeficiencies/imbalances, the results of ischemia and cell suicide. Thepresent invention may be used to treat all forms of spinal cord injury,including complete and incomplete injuries, ischemia, spinal cord injurywithout radiographic abnormality, central cord syndrome, anterior cordsyndrome, Brown-Séquard syndrome, posterior cord syndrome, tabesdorsalis and conus medullaris, among others.

The term “stroke” is used to describe a cerebrovascular accident (CVA),cerebrovascular insult (CVI), or brain attack, occurs when poor bloodflow to the brain results in cell death. There are two main types ofstroke: ischemic, due to lack of blood flow, and hemorrhagic, due tobleeding. Both of these types of stroke result in part of the brain notfunctioning properly. Signs and symptoms of a stroke may include aninability to move or feel on one side of the body, problemsunderstanding or speaking, a sense of spinning, or loss of vision to oneside, among others. Signs and symptoms often appear soon after thestroke has occurred. If symptoms last less than one or two hours it isknown as a transient ischemic attack. Hemorrhagic strokes may also beassociated with a severe headache. The symptoms of a stroke can bepermanent. Long term complications of stroke may include pneumonia orloss of bladder control The main risk factor for stroke is high bloodpressure. Other risk factors include tobacco smoking, obesity, highblood cholesterol, diabetes mellitus, previous transient ischemic attack(TIA), and atrial fibrillation, among others. An ischemic stroke istypically caused by blockage of a blood vessel. A hemorrhagic stroke iscaused by bleeding either directly into the brain or into the spacesurrounding the brain. Bleeding may occur due to a brain aneurysm. Bothischemic and hemorrhagic stroke are treated pursuant to the presentinvention.

The term “traumatic brain injury” (TBI) is used to describe an injury tothe brain caused by movement of the brain within the skill or an injuryto the brain caused by a foreign object. Causes of TBI may includefalls, a motor vehicle crash or being struck by or with an object. TBImay also be caused by a penetrating object—an injury to the brain causedby a foreign object entering the skull. Causes may include firearminjuries or being struck with a sharp object. TBI may cause aconcussion, a period of unconsciousness (coma) or amnesia. TBI mayimpair one or more of cognitive function (e.g., attention and memory),motor function (e.g., extremity weakness, impaired coordination andbalance), sensation (e.g., hearing, vision, impaired perceptin and touchand emotion (e.g., depression, anxiety, aggression, impulse control,personality changes).

The term “neurodegenerative disease” is used throughout thespecification to describe a disease which is caused by damage to thecentral nervous system and which damage can be reduced and/or alleviatedthrough transplantation of neural cells according to the presentinvention to damaged areas of the brain and/or spinal cord of thepatient. Exemplary neurodegenerative diseases which may be treated usingthe neural cells and methods according to the present invention includefor example, Parkinson's disease, Huntington's disease, amyotrophiclateral sclerosis (Lou Gehrig's disease), Alzheimer's disease, lysosomalstorage disease (“white matter disease” or glial/demyelination disease,as described, for example by Folkerth, J. Neuropath. Exp. Neuro., 58, 9,Sep. 1999), Tay Sachs disease (beta hexosamimidase deficiency), othergenetic diseases, multiple sclerosis, brain injury or trauma caused byischemia, accidents, environmental insult, etc., spinal cord damage,ataxia and alcoholism. In addition, the present invention may be used toreduce and/or eliminate the effects on the central nervous system of astroke or a heart attack in a patient, which is otherwise caused by lackof blood flow or ischemia to a site in the brain of said patient orwhich has occurred from physical injury to the brain and/or spinal cord.The term neurodegenerative diseases also includes neurodevelopmentaldisorders including for example, autism and related neurologicaldiseases such as schizophrenia, among numerous others.

The herein disclosed compositions, including pharmaceutical composition,may be administered in a number of ways depending on whether local orsystemic treatment is desired, and on the area to be treated.

Methods of treating subjects involve administration of a pharmaceuticalcomposition comprising an effective amount of EVs described herein andoptionally at least one additional bioactive (e.g. an agent which isuseful in the treatment of a neurodegenerative disease, stroke and/orspinal cord injury) agent. For example, the compositions could beformulated so that a therapeutically effective dosage of between about0.01, 0.1, 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90 or 100 mg/kg of patient/day or in some embodiments, greaterthan 100, 110, 120, 130, 140, 150, 160, 170, 180, 190 or 200 mg/kg ofthe disclosed EVs can be administered to a patient receiving thesecompositions.

The dose of EVs administered to a subject can be less than 10 μg, lessthan 25 μg, less than 50 μg, less than 75 μg, less than 0.10 mg, lessthan 0.25 mg, less than 0.5 mg, less than 1 mg, less than 2.5 mg, lessthan 5 mg, less than 10 mg, less than 15 mg, less than 20 mg, less than50 mg, less than 75 mg, less than 100 mg, less than 500 mg, less than750 mg, less than 1 g or more than 1 g. Administration may be bynumerous routes of administration, but intravenous, intrathecal,intranasal and/or intracerebrospinal fluid are often used as routes ofadministration.

In some embodiments, the disclosed EVs are administered within 24 aftera stroke or trauma. However, in some embodiments, the EVs areadministered at least 1, 2, 3, or 4 weeks after a stroke or trauma. Insome embodiments, the disclosed EVs are administered in multiple doses1, 2, 3, or more days apart. In some cases, such as cases ofneurodegenerative disease, the EVs are administered continuously (e.g.,once every 1, 2, 3, or 4 weeks) over the course of the disease.

EVs may be loaded with small molecules, antisense oligonucleotides,siRNAs, peptides, proteins or antibodies that target, peptides orpeptide translation products which are involved in neurodegenerativeprocesses.

In certain embodiments, the disclosed EVs are loaded with additionalbioactive agents or are co-administered with additional bioactiveagents, especially agents which are useful in the treatment ofneurodegenerative diseases.

The term “coadministered”, “coadministration” or “combination therapy”is used to describe a therapy in which at least two activecompounds/compositions in effective amounts are used to treat neuralinjury and/or a neurodegenerative disease. Although the termco-administration preferably includes the administration of EVs and atleast one additional active compound to the subject at the same time, itis not necessary that the compounds/compositions be administered to thepatient simultaneously, only that effective amounts of the individualcompounds/compositions be present in the patient at the same time. Thus,the term co-administration includes an administration in which the EVsand the bioactive agent(s) are administered at approximately the sametime (contemporaneously), or from about one to several minutes to abouteight hours, about 30 minutes to about 6 hours, about an hour to about 4hours, or even much earlier than the other compound/composition asotherwise described herein including up to a day or substantially more.

Agents which may be loaded or coadministered along with EVs may include,for example aricept, namenda, donepezil, excelon, razadyne, glantamine,rivastigmine, memantine, ergoloid, namzaric and mixtures thereof forAlzheimer's disease, biperiden, apomorphine, trihexyphenidyl,carbidopa/levodopa, rasagline, belladona, levodopa, benztropine,entacapone, selegiline, rivastigmine, pramipexole, rotigotine,bromocriptine, pergolide, ropinirole, carbidopa/entacapone/levodopa,amantadine, tolcopone, trihexiphenidyl and mixtures thereof, forParkinson's disease, tetrabenazine, haloperidol, chlorpromazine,olanzapine, fluoxetine, sertraline, nortriptyline, benzodiazpines,paroxetine, venlafaxin, beta-blockers, lithium, valproate,carbamazepine, botulinum toxin and mixtures thereof for the treatment ofHuntington's disease, anticholinergic drugs, anticonvulsants,antidepressants, benzodiazepines, decongestants, muscle relaxants, painmedications, stimulants and mixtures thereof for the treatment of motorneuron disease, selective serotonin reuptake inhibitors (SSRI's),selective norepinephrine-serotoning reuptake inhibitors (SNRI's),acetazolamide, baclofen, clonazepam, flunarizine, gabapentin, meclizine,memantine, ondansetron, scopolamine, modafinil, armodafinil, amantadine,atomoxetine, buproprion, carnitine, creatine, modafinil, armodafinil,pyrudistigmine, selegiline, venlafaxine, desvenlafaxine, buspirone,riluzole, verenicline, memantine, baclofen, tizanidine, cymbalta,lyrica, acetazolamide, carbamazepine, clonazepam, isoniazid, droxidopa,ephedrine, fludrocortisones, midodrine, levodopa, pramipexole,fluoxetine, n-acetylcysteine, baclofen, dantrolene sodium, diazepam,ropinirole, tizanidine, trihexylphenidyl, clonazepine, flunarazine,levetiracetam, primidone, topiramate, valproic acid, phenytoin,4-aminopyridine and mixtures thereof for the treatment ofspinocerebellar ataxia and riluzole for the treatment of spinal muscularatrophy. Agents for the treatment of stroke include salicylates, such asaspirin, a thrombolytic agent (alteplase) and a platelet aggregationinhibitor (clopidogrel), among others.

More generally, non-steroidal anti-inflammatory drugs (NSAIDS) and otheranti-inflammatory agents may be used in the treatment ofneurodegenerative diseases as described herein.

The activities of EVs described herein can be evaluated by methods knownin the art. The amount of EVs required for use in treatment can vary notonly with the particular cell from which the EVs are prepared, but alsowith the route of administration, the nature of the condition beingtreated and the age and condition of the patient and can be ultimatelyat the discretion of the attendant physician or clinician. In general,however, a dose can be in the range of from about 0.01 mg/kg to about 10mg/kg of body weight per day.

Identifying EVs useful in the present methods for treating a spinal cordinjury, stroke, traumatic brain injury and/or a neurodegenerativedisease which occurs by modulating the activity and expression of adisease-related protein and biologically active fragments thereof can bemade by screening EV activity in any of a variety of screeningtechniques. The screening can be made for whole EVs or their contents.Fragments employed in such screening tests may be free in solution,affixed to a solid support, borne on a cell surface, or locatedintracellularly. The blocking or reduction of biological activity or theformation of binding complexes between the disease-related protein, theEVs and/or one or more components of the EVs may be measured by methodsavailable in the art.

A number of embodiments of the invention have been described.Nevertheless, it will be understood that various modifications may bemade without departing from the spirit and scope of the invention.Accordingly, other embodiments are within the scope of the followingclaims.

EXAMPLES

Studies were conducted to determine whether EVs confer paracrinebenefits on neural stem cells and play a role in both optimal in vitroneural cultures conditions and in therapeutic outcomes of neural stemcell treatments. The objective of the study was to determine if it waspossible to isolate EVs from human stem cells of a neural lineage,specifically neural progenitor cells and/or differentiated post-mitoticneuronal cells. EVs were purified from neural progenitor cells (SOX 1+and 2+, OCT4-; hNP1™ ArunA Biomedical), derived from human pluripotentstem cell lines or differentiated neuronal cells (β-III tubulin (Tuj1)+, MAP2+, Oct4-); hN2™ ArunA Biomedical).

Example 1

Human pluripotent stem cells [see, Chambers, et al., Methods Mol Biol,2016. 1307: p. 329-43] lines were cultured in media absent of serum suchas MTeSR or E8 which is commercially available from vendors such StemCell Technology using feeder free conditions or media composed ofknock-out serum replacement (KSR) media (DMEM=F12, 2 mM L-glutamine, 0.1mM MEM nonessential amino acids, 50 U/mL penicillin, 50 mg/mLstreptomycin, and 20% KSR) (all from Gibco, Carlsbad, Calif.) and 4ng/mL basic fibroblast growth factor bFGF; R&D Systems, Minneapolis,Minn.). Cells were cultured on Mitomycin-C (Sigma, St. Louis, Mo.)mitotically inactivated murine embryonic fibroblasts (MEF) or withoutfeeders, manually or enzymatically dissociated, and passaged to newfeeder layers every 2-5 days. For feeder-free culture of hPSC inConditioned media, cells grown on MEF were washed once with PBS (withoutCa2+ and Mg2+), and then incubated with 0.25% trypsin (Gibco) until theMEF layer began to lift off the dish. The floating MEF layer wasdiscarded after agitating it to release adherent stem cells that werecollected, centrifuged, and resuspended in MEF-conditioned media (CM).CM was prepared by placing 20% KSR media on MEF for 24 h and thensupplementing the collected media with an additional 4 ng/mL of bFGF.Cells were plated on tissue culture dishes coated with laminin substrate(1 mg/cm2; Sigma) and grown to −90% confluence. The cells were passagedat least three times to minimize MEF contamination [see, Boyd, et al.,Tissue Eng Part A, 2009. 15(8): p. 1897-907, Mumaw, et al., MicroscMicroanal, 2010. 16(1): p. 80-90 and Young, et al., Neuroscience, 2011.192: p. 793-805,]. Regardless of the culture method all media iscollected and EVs are collected from the media and used to treatpatients

After manual passage onto fresh feeder cells, hESCs were allowed toproliferate in ES medium for 7 days (stage 1). Cell differentiation wasthen induced with either DN2, MEDII, or ES medium for another 7 days(stage 2). DN2 medium is DMEM/F12-based medium supplemented with N2(Gibco), L-glutamine, penicillin/streptomycin (P/S), and 4 ng/ml bFGF.MEDII medium for this study is DN2 medium supplemented at 50% (unlessotherwise noted) with conditioned medium. To understand and follow thedifferentiation steps applied here, phenotype marker expression wasexamined at various time intervals. At stages 1, 2, and 3, populationswere harvested and the markers Musashi-1, Nestin, and Oct-4 wereobserved. Immunocytochemical analysis was also performed on the adherentcell population. The cells at both stages were double-stained withNestin and Oct-4 and observed under the fluorescence microscope forimmunocytochemical examination associated with morphology. Groups thatdisplayed phenotypic difference were then subjected to quantitativeanalysis for these same markers using flow cytometry. All experimentswere replicated three times unless otherwise noted. After manual passageonto fresh feeder cells, hESCs were allowed to proliferate in ES mediumfor 7 days (stage 1). Cell differentiation was then induced with eitherDN2, MEDII, or ES medium for another 7 days (stage 2). DN2 medium isDMEM/F12-based medium supplemented with N2 (Gibco), L-glutamine,penicillin/streptomycin (P/S), and 4 ng/ml bFGF. MEDII medium for thisstudy is DN2 medium supplemented at 50% (unless otherwise noted) withconditioned medium (described above). Stages 1, 2, and 3, populationswere harvested and the markers Musashi-1, Nestin, and Oct-4 wereobserved. Immunocytochemical analysis was also performed on the adherentcell population. The cells at both stages were double-stained withNestin and Oct-4 and observed under the fluorescence microscope forimmunocytochemical examination associated with morphology. Groups thatdisplayed phenotypic difference were then subjected to quantitativeanalysis for these same markers using flow cytometry. All experimentswere replicated three times unless otherwise noted.

After manual passage onto fresh feeder cells, hESCs were allowed toproliferate in ES medium for 7 days (stage 1). Cell differentiation wasthen induced with either DN2, MEDII, or ES medium for another 7 days(stage 2). DN2 medium is DMEM/F12-based medium supplemented with N2(Gibco), L-glutamine, penicillin/streptomycin (P/S), and 4 ng/ml bFGF.MEDII medium for this study is DN2 medium supplemented at 50% (unlessotherwise noted) with conditioned medium (described above). At stages 1,2, and 3, populations were harvested and the markers Musashi-1, Nestin,and Oct-4 were observed. Immunocytochemical analysis was also performedon the adherent cell population. The cells at both stages weredouble-stained with Nestin and Oct-4 and observed under the fluorescencemicroscope for immunocytochemical examination associated withmorphology. Groups that displayed phenotypic difference were thensubjected to quantitative analysis for these same markers using flowcytometry. All experiments were replicated three times unless otherwisenoted.

hPSC were allowed to proliferate in hPSC medium (any of those describedabove) for approximately 5 to 10 days (stage 1) as a monolayer or asembryoid bodies. Cell differentiation was then induced with mediumtypically DMEM/F12-based medium supplemented with N2 (Gibco),L-glutamine, penicillin/streptomycin (P/S), and usually 4 ng/ml bFGF.Nestin, and Oct-4 were observed and neural rosettes form. Neuralrosettes are isolated (manually or enzymatically and areimmunocytochemical analysis was also performed on the adherent rosettepopulation. The cells at both stages were double-stained and are Nestin+and Oct-4− and observed under the fluorescence microscope forimmunocytochemical examination associated with morphology [Shin, et al.,Stem Cells, 2006. 24(1): p. 125-38]. The rosettes when isolated frommost of the contaminating cells are considered human neural progenitorcells. Briefly, hNP cells were grown on poly-ornithine (20mg/mL)/laminin (Sigma-Aldrich, Inc.) (5 mg/mL) coated plates or otherECM such as Matrigel, and maintained and expanded in media with 2 mML-glutamine and 20 ng/mL bFGF. hNP Cells were passaged approximatelyevery 48 h and split 1:2 following manual dissociation [see, Mumaw, etal., Id., Young, et al. Id. and Dhara, et al., Methods Mol Biol, 2011.767: p. 343-54].

Alternatively, neural induction may be stimulated by inhibition of SMADsignaling using inhibitors of Activin/Nodal pathway, and/or BMPsignaling (examples of inhibitors might be Noggin, SB431542 [Chambers,et al. Nat Biotechnol, 2009. 27(3): p. 275-80], Compound C [Zhou, etal., Stem Cells, 2010. 28(10): p. 1741-508], or other strategies aloneor in combination). Cells may be cultured on matrigel or otherextracellular matrices, in AB2, Neurobasal, or other mediums listedabove, in the presence of absence of Sonic Hedgehog [Chambers, et al.,Id., and Zhou, et al., Id.].

hNP cells differentiated into neurons on poly-ornithine and laminincoated plates or other ECM such as Matrigel under maintenance mediadescribed above without bFGF and LIF. Alternatively LIF or EGF are addedhNP cells were allowed to differentiate under these two conditions for 1to 7 weeks [Mumaw, et al., Id., and Dodia, et al., PLoS One, 2011. 6(8):p. e232669].

After 1 to 4 weeks of neuronal differentiation such as hNP cellsdifferentiated into neurons on poly-ornithine and laminin coated plates(under two conditions in maintenance media without bFGF or bFGF and LIFor EGF), astrocytic differentiation was induced by switching the neuralto cells from hNSC Maintenance Medium to the basic medium (DMEM HAM'sF12 medium, glutamine, Penicillin/Streptomycin) supplemented with 1% FCS(Gibco). See Mumaw, et al., Id. and Young, et al., Id. Finally,multilinear differentiation was achieved by replacing the maintenancemedium by the basic medium containing 10% of FCS. After 45-50 dayscultures were nearly 100% positive for glial markers S1000 and vimentin[Palm, et al., Sci Rep, 2015. 5: p. 1632110].

Alternatively, hNP cells were propagated as adherent monolayer culturesin a proliferation media (such as AB2™, ANS™ Neurobasal™, 1×B27, 1×Glutamax™, P/S, FGF2 (10 ng/mL) as described (Shin et al., Id.), anddifferentiated by removal of FGF. For astrocytic differentiation of hNPcells, neuronal differentiation media were supplemented with recombinantproteins, such as BMP2 and combinations of chemicals such asAzacytidine, Trichostatin A, or similar molecules for 1-5 days, withcomplete media changes in between, followed by differentiation mediasupplemented with the molecules separately or in combination. Cells wereharvested prior to analysis at 5, 15 or 30 days of treatment or forcryopreservation at d6 or d10 of differentiation. For cryopreservation,cells were dissociated with Accutase™ and frozen in differentiationmedia containing 10% DMSO [Majumder, et al., Stem Cell Res, 2013. 11(1):p. 574-86].

When hPSC cultured without feeders as described above reachedapproximately 90% confluence, the 100 mm dishes were washed withPBS++(with Ca2+ and Mg2+) and replaced with 10 mL of fresh endothelialgrowth media 2 microvascular (EGM2-MV) (Lonza; 5% FBS, proprietaryendothelial basal media 2 (EBM2) basal media and concentrations of bFGF,VEGF, EGF, and R3-IGF-125). The media was changed every 2-3 days over aperiod of 20-30 days. After transition from hESC to epithelial sheet wascompleted, the cells were trypsin passaged to a T75 flask and grown toconfluence. To expand the initial cell culture, cells were passaged andseeded at a target density of approximately 4×10⁴ cells/cm2 per flasks.For subsequent culture for experimentation, cells were subcultured at10⁶ cells=T75 flask (approximately 1.3×10⁵ cells/cm2) and grown toconfluence over 5-7 days [Boyd, et al., Id].

Example 2

Methods

Cell medium was collected from confluent cultures 24 hours post mediachange and frozen at −20° C. Medium was thawed at 4° C. overnight andfiltered through a 0.22 μm Steriflip unit prior to EV purification.

Extracellular vesicle purification by ultracentrifugation.

Isolation of extracellular vesicles from cell culture media wasperformed according to the protocols published by Théry, C., et al.,Isolation and Characterization of EVs from Cell Culture Supernatants andBiological Fluids, in Current Protocols in Cell Biology. 2001, JohnWiley & Sons, Inc. Briefly, filtered media was serially centrifuged at300×g for 10 minutes, and supernatant was transferred to a fresh tubefor centrifugation at 2,000×g for 10 minutes. Collected supernatantswere then centrifuged at 10,000×g for 30 minutes, and resultantsupernatant collected into a fresh tube. To label EVs, DiI was added tothe purified supernatants at a final concentration of 10 μM andincubated at room temperature for 30 minutes. Supernatants weredistributed into 11.5 ml Sorvall Ultracrimp tubes and sealed beforetransfer into a Sorvall T880 fixed angle rotor for centrifugation at100,000×g for 70 minutes at 4° C. Supernatant was carefully removed andpelleted material resuspended in PBS and transferred into anotherultracrimp tube, and again centrifuged at 100,000×g for 70 minutes at 4°C. The PBS was removed and pelleted material from each tube wasresupended in 100 μl PBS. All purified EVs from the same cell type werepooled, triturated, and aliquoted into DNase/RNase free tubes (20-50 μlaliquots) for storage at −20° C.

Extracelluar Vesicle Purification by Ultrafiltration

Ultrafiltration of extracellular vesicles was performed according to theprocedure developed for purification of cardiomyocyte derivedextracellular vesicles. Amicon Ultra-15 100 kDa molecular weight cutofffilters were wetted with 10 ml PBS and centrifuged in a swinging bucketrotor at 4,000×g for 10 minutes. The PBS was discarded and the cellculture medium was added to the filter approximately 15 ml/tube andcentrifuged at 4,000×g for 10 minutes. Another 15 ml culture medium wasadded to the filter when filtering stem cell derived extracellularvesicles, because less media was retained in the filter from the firstrun through, so approximately 30 ml of medium total was filtered for theH9 derived NP, Astrocyte, and MSC lines, while only 15 ml of medium wasfiltered for SH SY5Y cells; the media in the filter was centrifuged at4,000×g in 5 minute increments to obtain approximately 1 ml ofretentate. This was then either moved to a 1.5 ml tube for DiI labeling(at 10 μM for 30 minutes) or for unlabeled purification diluted to 15 mlwith PBS and washed twice before repeating 5 minute centrifugationincrements until a final volume of 1-1.5 ml was obtained. The purifiedextracellular vesicle preparation was then pooled from the same celltype and dispersed into approximately 100 μl aliquots (DNase/RNase freetubes) and stored at −20° C.

DiI-Labeling Vesicles

Labeled vesicles were generated by ultrafiltration. After theultrafiltration was complete, the filter retentate was moved to acentrifuge tube, and 10 μM DiI was added to the supernatant for 30 min.PBS was added to the filtration unit during this time to prevent thefilter from drying out. After incubation with the labeling agent,supernatant was transferred back into the filtration device and washedthree times with PBS (approximately 45 ml) to remove free label. Afterthe final wash the retentate was concentrated to approximately 1 ml,which was aliquoted and stored at −20° C.

Electron Microscopy

Vesicle preparations were mixed 1:1 with 4% paraformaldehyde (PFA) (toyield 2% PFA final) and incubated for 15 minutes. 5 μl droplets of fixedvesicle suspensions were transferred to Formvar-coated grids for 20min., and then washed by transferring to drops of PBS. Grids weretransferred onto drops of 1% glutaraldehyde for 5 min, and then movedover several drops of water to remove residual glutaraldehyde beforetransferring to uranyl-oxylate. Grids were imaged by electron microscopyat 80 kV.

Results

While initial reports indicate that neural progenitor cells secretefewer EVs than other cell types, ultrastructural analysis of the cellsrevealed prominent vesicles of endocytic origin (FIG. 2A-2C), and manywere in close proximity or associated with the outer limiting membraneof the cell (FIG. 2D, 2E). Cargoes within the multivesicular bodies(MVBs) vary in size (FIG. 2 B, 2C) and are occasionally visualizedbudding off into the vesicle (arrow). Smaller vesicles seem to coalesceinto larger MVBs as they move to the periphery of the cell (FIG. 2F,2G). It was possible to purify and visualize vesicles from the media ofneural progenitor cells (FIG. 2H). These data suggest that neuralprogenitor cells do release extracellular vesicles into the cellularmedium, and these vesicles can be purified using published EVpurification protocols. All scale bars are 500 nm.

After realizing that neural progenitor cells have the capability ofproducing EVs that could be purified and visualized by electronmicroscopy, the process of characterizing the purified vesicles frommultiple cell types began with protein profiling by Coomassie stain(FIG. 3A), and BCA Analysis of total protein content (FIG. 3B). Theprotein profiles were compared from neural progenitor cells,differentiated neural cells, and astrocytes, all derived from the sameES cell line, and SH SY5Y cells, a human neuroblastoma cell line used asa positive control. Early experiments indicate that the protein profileoverlaps, but there are distinct proteins in the neural progenitor anddifferentiated neural cells, even though the cells are of the samegenetic origin. Similarly, comparing the size profiles of the vesiclesfrom neural progenitors, and astrocyte cells from the same geneticorigin indicates that the size of vesicles from the 2 cell types overlap(including a large percentage of 55 nm vesicles), but there are distinctsubpopulations that vary between the two cell types, with astrocytesshowing unique vesicle sizes including a 25 nm population, and slightlylarger 135 nm population (FIGS. 3C, 3D). Taken together, these dataindicate that cargoes are specifically targeted into MVBs, and thesecargoes change in the differentiation process, supporting a role for EVsin cell to cell communication throughout the process of development.

In order to determine if it was possible to label EVs, and visualizetheir uptake by another cell, differentiated neural cells (6-8 weeks indifferentiation medium without fibroblast growth factor (FGF) weretreated with astrocyte derived vesicles at timepoints ranging from 30seconds through 30 minutes, after which, cells were fixed, and stainedfor f3-III tubulin. At the earliest time points, few vesicles were foundin the cells, (FIGS. 4A-4C). By 5 minutes in the culture it was possibleto find neural cells with prominent red fluorescence, indicating vesicleuptake in these cells (FIGS. 4D-4F).

To determine if these vesicles could elicit an effect in recipientcells, differentiated neural cells were treated with serially dilutedconcentrations of vesicles derived from neural progenitors, astrocytes,or MSCs all derived from the same ES cell line, or SH SY5Y cells. Thesecells were then subjected to nutrient deprivation over 10 days andanalyzed for neurite outgrowth. As expected, in wells that received onlyPBS the monolayer was disrupted and few cells were left (FIG. 5. D).However, cells that received the highest concentrations of EVs fromeither neural progenitors or astrocytes were able to survive thisnutrient deprivation, and the monolayer of cells with intact neuriteswere largely still intact (FIG. 5. A, B). Wells treated with MSC derivedEVs contained more cells than untreated wells, but the monolayer was nolonger intact and neurites were largely lost (FIG. 5. C). These dataindicate that vesicle treatment protected the recipient cells fromnutrient deprivation, and importantly, indicate that cells responddifferently to vesicles that originate from different cell types, evenif the vesicles are from an isogenic or autologous source.

Taken together, these data support the idea that the parental source oforigin has an impact on the vesicles that result. This has hugeimplications for considering extracellular vesicles as a therapeuticsource that can potentially be exploited for regenerative medicine, andhighlights the need for vesicles derived from neural sources for thetreatment of CNS injuries and/or disease. Importantly, these data alsoindicate that not only neuron derived vesicles, but also glial vesiclesprovide benefit in vitro.

Example 3

Biodistribution Methods: Rodent biodistribution by single-photonemission spectroscopy. 1.5-2 mCi of Indium-111-oxine in PBS was added to200 μl doses containing EVs (˜2.7×10¹¹ vesicles/kg) and incubated at 37°C. for 20 minutes. Free indium was removed by three repeated PBS washesthrough an Amicon 100 kDa ultrafiltration device. Collected EVs werediluted to 200 μCi of radioactivity per dose, and injected intravenouslyinto the mouse tail vein, either 1 hour or 24 hours post-stroke. Controlanimals received injection of free indium-111-oxine. Whole body and headsingle photon emission spectroscopy (SPECT) images were acquired byMediso's nanoScan microSPECT/CT system 1 and 24 hours after injection,and projection images were reconstructed according to maximum intensityto determine radioactivity in the brain and throughout the body.

Piglet biodistribution: EVs were concentrated by ultrafiltration usingCentricon units as previously described and then moved to 50 ml falcontubes for DiR labeling (5 μM) in the dark for 30 minutes. Labeled EVswere collected by ultracentrifugation at 100,000×g for 4 hours. PelletedEVs were washed with PBS and again collected by ultracentrifugation. EVswere resuspended in PBS and diluted into 2.7×10¹⁰ vesicle/kg body weight(in 200 ul PBS) doses for individual piglets based on NanoSightNS 300nanoparticle tracking analysis. Piglets were anesthetized withisoflourane for intravenous injection (tail vein), intranasal delivery,cerebrospinal fluid EV injection into the subarachnoid cistern, orinjection directly into the brain parenchyma. For intraparenchymalinjection EVs were delivered at a flow rate of 5 μl per minute. Animalswere euthanized by isoflourane followed by CO₂ asphyxiation 30 minutesafter completion of EV delivery. Brain, heart, liver, kidneys, lungs,and spleen were removed and imaged using Lumina IVIS (model) to detectDiR fluorescence.

Mouse: EV biodistribution was evaluated after either free indium-111 orindium-111 labeled EVs were injected into the mouse tail vein eitherimmediately (FIG. 6B), or 24 hours post-stroke (FIG. 6C). SPECT scanswere performed 1 hour after injection (FIG. 6A, 6B, 6C, left panels),and again 24 hours after injection (FIG. 6A, 6B, 6C, right panels).Images reconstructed by maximum intensity indicate that indium-111labeled EVs are distributed throughout the bodies filtration organs inlungs, liver, spleen, and kidneys at both the initial timepoint, and 24hours later (FIG. 6B, 6C; whole body scans) while free indium-111 isinitially localized mostly to the lungs before disbursing into theliver, spleen, and kidneys 24 hours later (FIG. 6A; whole body scans)likely indicating clearance through the renal system. Uniquely, labeledEVs were present in the brain in proximity of the stroke within 1 hourof injection when injected either immediately or 24 hours after stroke(FIG. 6B, 6C; circles), indicating either access from the circulationdue to disruption of the blood brain barrier, homing to the damagedtissue, or some combination of the two. Free indium did not localize tothe stroked tissue at either time point assessed (FIG. 6A, circles).

Uniqueness: This is the first time that anyone has shown that IVinjection of EVs are actually distributed in and around the infarctedbrain following stroke. The majority of the EVs were distributed inother organs such as heart, liver, lung, and kidney, and we have data tosuggest that the EVs for the first time have a systemic effect on theimmune response post stroke (data in other sections). Thus it is likelythat systemic effects of peripheral EVs have a positive effect onmolecular and phenotypic benefits following stroke. Thus the EVs mayhave a direct effect at the site of injury, could be via local immunecells or via direct effect on the neurons, as well as a system effect onthe immune system. This is the first data showing biodistribution in alarge animal brain.

Piglet: In order to optimize EV delivery to the brain following stroke,biodistribution of fluorescently labeled (DiR, 5 μM) was evaluated inuninjured piglets. Labeled EVs were detected in the brain afterinjection directly into the parenchyma (FIG. 7, top panels), or into theCSF of the subarachnoid cistern at the base of the skull (FIG. 7, lowerpanels). This is the first demonstration of EV biodistribution in alarge animal study.

Example 4

Methods

Mouse Model

Middle-aged C57/B6 male mice (retired breeders of 9-11 months) werepre-trained for adhesive tape test (ATT) for 3 days prior to the strokesurgery (3 trials/day). Mice were marked for identity, numbered, andrandomized for therapy after stroke in a block size of 4 (4 animals fromthe same cage) to different treatment groups following induction ofembolic stroke. The surgeon performing the stroke surgery and cerebralblood flow (CBF), and the investigator performing neurobehavior andneurologic deficit scoring remained blinded to the identity of thegroups. All four therapies received were thawed and injectedintravenously at 2, 14 and 38 hrs post stroke with doses 1, 2 and 3,respectively. Relative cerebral blood flow (CBF) was measured at 6 and48 hrs post stroke. Neurologic deficit score (NDS) was assessed at 48hrs post stroke; the ATT that reflects the somatosensory function wasperformed at 96 hrs post stroke just prior to euthanasia. Mortality wasmonitored daily and recorded until day 4 and prior to euthanasia.

Embolic Stroke

Mice were sedated with Buprenorphine (0.05 mg/kg SC) 20 min prior tostroke surgery, and anesthetized with 3.5% isofluorane. Surgical plainof anesthesia was maintained with 1.5-2.0% during the surgery. Bodytemperature was maintained at 37° C. by a thermo-regulated surgery pad.By a midline incision on the ventral side of the neck, the right commoncarotid artery (CCA), external carotid artery (ECA), and the internalcarotid artery (ICA) were assessed. A temporary atraumatic clip wasplaced on the CCA to prevent loss of blood during catheter insertion. Amodified PE-10 catheter containing a single fibrin rich clot (9±0.5 mmlength) was introduced into the ECA and advanced into the ICA. The clotwas gently injected with 100 μL of PBS, the catheter was removedimmediately after embolization, and the arterial wound was secured toprevent blood loss. Induction of stroke was confirmed using on-siteportable single point cortical laser Doppler flowmetry (PeriMed Inc.).Finally, the temporary clip was removed and the blood flow in the CCAwas reinstated. The site of surgery was closed using #6 sterilemonofilament nylon suture, and Buprenorphine (0.05 mg/kg SC) was againinjected. Mice were transferred to clean recovery cages and animaltemperature was maintained. Conscious mice were transferred to cleanregular cages with free access to food and water. NAPA gel and lactatedringers solution were provided as needed, in case of any sign ofdehydration; otherwise 1 ml of regular sterile saline pre-warmed at 37°C. was injected SC every 12 hours.

Laser Speckle Contrast Imaging:

Briefly, 6 hrs after stroke, mice were anesthetized using isofluorane,while body temperature was maintained at 37±0.5° C. The skull was shavedand a midline skin incision was made to expose the middle cerebralregion. Perfusion images were acquired using PeriCam high resolutionLaser Speckle Contrast Imager (LSCI; PSI system, Perimed) with a 70 mWbuilt-in laser diode for illumination and 1388×1038 pixels CCD camerainstalled 10 cm above the skull (speed 19 Hz, and exposure time 6 mSec,1.3×1.3 cm). Acquired video and images were analyzed for dynamic changesin CBF. Overall perfusion of the ischemic region will be compared to anequally sized region of interest from the uninjured contralateralhemisphere to estimate relative CBF. The skin wound was closed usingtissue glue. At 48 hrs post-stroke, the skin wound was again opened,cleaned, and the middle cerebral artery region was exposed to repeat theLSCI procedure as previously described.

Neurologic Deficit Score (NDS):

Neurologic deficits in mice were assessed by investigators blinded tothe therapeutic group at 48 hr post stroke on a 5-point scale with thehighest number indicating the worst outcomes and lower number indicatingbetter neurological outcomes according to the following criteria: 0, nodeficit (normal mice); 1, forelimb flexion deficit on contralateralside; 2, flexion deficit along with decreased resistance to lateral pushand torso turning to the ipsilateral side when held by tail; 3, Alldeficits as in Score 2, including very significant circling to theaffected side during the move inside the cage, and reduced capability tobear weight on the affected side; 4, All deficits as above but rarelywilling to move spontaneously, and prefer to stay in rest; 5, consideredterminal and euthanized in accordance with animal care requirements.

ATT for Somatosensory Test:

Adhesive tape test (ATT) was used as a test of somatosensory motorfunction, and was performed at 96 hrs post-stroke immediately prior toeuthanasia. Briefly, naïve mice were acclimatized to the procedure ofthe test for 3 days prior to surgery by placing them in a transparentacrylic box (15 cm×25 cm). Two pieces of adhesive tape (0.3 cm×0.4 cm)were used as bilateral tactile stimuli after they were attached at thedistal-radial region on each forelimb such that it covered the hairlesspart (3 pads, thenar and hypothenar). Within 180 seconds, the taperemoval time was recorded as the sensorimotor function. If a mousefailed to remove the tape within 180 seconds, it was given a score of180 seconds. Therefore, a shorter time score indicates a better outcomewhile longer time indicates an outcome with higher deficit.

Euthanasia and 2,3,5-Triphenyltetrazolium Chloride (TTC)-Staining:

TTC-stain differentiates between metabolically active (live or penumbra)and inactive (dead or core) tissues after stroke. TTC is a colorlesssolution, which is reduced to red 1,3,5-triphenylformazan (TPF) by theenzymatic action of various dehydrogenases primarily mitochondrialdehydrogenase from the living tissues, while the core (lead tissue)remains white. Therefore, larger white area indicates higher injury andinfarction volume. At 96 hrs post stroke and after performing ATT, micewere deeply anesthetized with isofluorane (5%). Blood was collected viadirect cardiac puncture to isolate and obtain serum later. Brains werevery quickly perfused with 25 mls of cold 0.01 M phosphate-bufferedsaline (PBS), harvested fresh, and immediately transferred to a metallicmouse brain matrix. Looking at the infarcted area, 5-blades were placedin alternate gaps to obtain 2-mm coronal slices (4 sections per brain).Sections were individually placed in a 35-mm dish containing pre-warmed(37° C.) 3 ml of 5% TTC in PBS (Sigma) for 20-30 minutes at 37° C.,followed by 2× washing with cold PBS and fixation with 10% formalin. Inorder to image, fixed sections were taken out of the dish and placed inorder on a high-resolution Cannon Scanner. Images were cropped and savedfor analysis. Corrected infarct volume was estimated using gray scaleimage and Scion Image software, and presented as the % volume of theuninjured side.

Blood Sample Flow Cytometry (Th17, Treg, M2)

Prior to euthanasia blood was collected and purified cells weresubjected to fluorescence activated cell sorting to identify populationsof immune cells present systemically including T-helper (CD4+/FOX3P+)populations, regulatory T-cells (CD4+/IL17+), and M2 macrophage(IL10+/CD206+) populations.

Results

EVs were purified from isogenically derived astrocyte progenitors(APEX), MSCs (MSCEX), and neural progenitor (NPEX) cells using standardmethods (see manufacturing section), evaluated by nanoparticle trackinganalysis (NanoSight-NS300) and stored in individual dose aliquots at−20° C. until they were thawed at room temperature immediately prior totail vein injection following embolic stroke. The 3 EV types and PBSwere administered by blinded investigators following embolic stroke, in3 doses of approximately 2.7×10¹¹ vesicles/kg (or vehicle) at 2, 14, and28 hours post-stroke. In all parameters evaluated both APEX and NPEXoutperformed the vehicle treated controls as well as the MSCEX treatmentgroup. Immediately after euthanasia, 2,3,5-Triphenyltetrazolium Chloride(TTC) was used to differentiate metabolically active (live, red) andinactive (dead, colorless) tissues. Importantly, TTC indicates decreasedinjury and infarction volume following APEX or NPEX™ treatment (FIG. 8A), while MSCEX treatment was comparable to control. Within the 96 hrsfollowing stroke APEX and NPEX treatments decreased mortality by 20 and17% respectively (FIG. 8B). Neurologic deficits evaluated 48 hrs poststroke (on a 5-point scale from no deficit [0] with increasing severitythrough terminal deficits [5]) indicated significantly better behavioraloutcomes for mice that received APEX or NPEX (FIG. 8D). Ability toremove adhesive tape used as bilateral tactile stimuli attached at thedistal-radial region of each forelimb indicated improved sensorimotorfunction as well (FIG. 8E). Taken together, these data indicate improvedsurvival, molecular, and functional outcomes in NPEX treatedthromboembolic stroke rodent models compared to contemporary vehiclecontrols.

Flow cytometric analysis of blood cells at the 96 hour time point justprior to euthanasia indicates that neural cell type derived EVs, APEXand NPEX treatment resulted in an increase in the presence of protectiveregulatory T cells in circulation (FIG. 9A), while MSCEX wereindistinguishable from controls. Pro-inflammatory T-helper cells werereduced in the APEX and NPEX groups (FIG. 9B), while anti-inflammatoryM2 macrophages were increased in these groups (FIG. 9C). Taken together,these data indicate that neural cell type derived EVs exert part or allof their effects by modulating the immune response following stroke.

The production and quality control methods used to produce isogenically(genetically identical) derived neural progenitor, astrocyte progenitor,and MSC cells from the same ES cell line produce a unique opportunity tocompare the EVs from these 3 cell types without the confounding variableof genetic variation due to source of the donor material. The vastmajority of stem cell derived EV literature centers on use of MSCderived EVs. Here, for the first time neural cell derived EVs (APEX andNPEX) were evaluated and compared directly with MSC-derived EVs. Thereis a substantial improvement in molecular benefit (infarct volume), alsoincreased survival and improved functional outcomes of neural derivedEVs (APEX and NPEX) over the MSCEX. These improvements were immediate inolder animals which is more prone to death. Thus no other group hasshown that any EV (MSC or neural) therapy has such stark and immediateimprovements in a mouse model that replicates the human stroke condition(embolic stroke) and factors in co-morbidly factors such as age (middleage mice). It appears that these early strong effects can only beobtained with a neural EV as disclosed herein.

These studies suggest that the mechanism of action in part may bethrough immune modulation suppressing the inflammatory M1 responseincluding but not limited to suppressing IL17 cytokine while enhancingthe M2 response perhaps through IL10 or other cytokines.

Example 5

Pig Model, ischemic injury induced by middle cerebral artery (MCA)occlusion.

Landrace barrows (5-6 months, 150-170 lbs) were subjected to injury aspreviously described using the only fully developed porcine stroke model[1]. Briefly, a portion of the zygomatic arch was resected and theunderlying muscle was elevated dorsally revealing the parietal bone. Awindow was generated in the bone surface exposing the dura mater. Theproximal MCA was permanently occluded resulting in infarction spanningthe most caudal aspect of the frontal lobe as well as significantportions of the temporal, parietal, and occipital lobes.

Magnetic Resonance Spectroscopy

Magnetic resonance imaging (MRI) was performed 24 hours and 90 dayspost-MCAO surgery on a GE 16-channel fixed-site Twin gradient Signa HDx3.0 Tesla MRI system. Under anesthesia, MRI of the neurocranium wasperformed using a multichannel phase array spine coil, with the patientin dorsal recumbency. Standard multiplanar MR brain imaging series wereacquired. These included T2-weighted (T2w), T2-weighted fluid attenuatedinversion recovery (FLAIR), and T1-weighted (Tlw) FLAIR, as well asdiffusion-weighted imaging (DWI) series. DWI was acquired with b=0 andb=1000. DWI, apparent diffusion coefficient (ADC) maps and Tlw-FLAIRimages were evaluated using Osirix® software for presence of cerebralinfarction and changes in cerebral hemisphere volume. Specifically, thevolume of the ischemic area was manually derived from the ADC mapsgenerated from DWI sequences. The ischemic area was defined by twolevels of ADC number reduction, with the ADC number from thecontralateral cerebral hemisphere providing normal ADC values. Ischemicareas, defined by those with 80% and 40% ADC values of normal, weremanually traced on the sequential ADC images. Each area was multipliedby slice thickness to produce a volume of ischemic tissue. This methodwas chosen because it has been demonstrated to strongly correlate withhistologically defined areas. The cerebral hemisphere volume wasdetermined through a similar process, whereby the cerebral hemispherevolume was quantified (excluding sulci and the lateral ventricularspaces) on Tlw FLAIR images. The T2w FLAIR images were used forreference to differentiate areas filled with CSF and parenchymal areasof hyperintensity (Platt, S. R., et al., Experimental & TranslationalStroke Medicine, 2014. 6:5).

EV Dosage and Administration

NPEX EVs containing ˜2.7×10¹⁰ vesicles/kg in 50-60 ml PBS were thawed at4° C. and transferred into a 60 cc syringe using aseptic technique whilein a biological safety cabinet. Samples were inverted a minimum of 25times immediately prior to intravenous injection via an ear veincatheter. Pigs received either 3 doses (50-60 ml) of NPEX or PBS(vehicle) at 2, 14, and 24 hours post-MCAO.

Animal Evaluation and Recovery

Following surgery, animals were moved to a clean recovery pen andmonitored continuously until extubation. Rectal temperature, heart rate,and respiration rate (TPR) were recorded every 15 minutes until the pigwas awake and vital signs were stable within normal limits. Thereafter,TPR measurements were initially reduced to 1-2 hour intervals unlessvital signs deviated from normal (for example, a fever), then to longerintervals over the next 48 hours as the pig recovered. During the first36 hours, pigs were never left for more than 4 hours withoutobservation, and generally for no more than 2 hours. In addition to TPR,other observations recorded included time from arrival in recovery untilthe animal first stood up on its own, time to drink and eat withassistance, and time to eat and drink unassisted. Events such as fevers(rectal temperature 103° F. or greater), circling behavior, and seizureswere also monitored and documented.

As seen in Table 1, routine evaluation indicates improved survival andfunctional outcomes in immediate recovery post-stroke with NPEXtreatment. Survival was substantially better in the NPEX treatmentgroup, with 7/7 pigs surviving more than 72 hours post-stroke, whileonly 5/8 survived in the control group. Time until the animals couldstand unassisted was reduced by about 2 hours with NPEX treatment.Fevers, which are very common within 72 hours post-stroke, were reducedin the NPEX treatment group where 4/7 animals had one or more feverepisodes as compared to control group where 6/8 animals had at least onefever. Time to eat and drink unassisted, number of animals exhibitingcircling behavior, and number of animals with documented seizureactivity were similar between groups.

TABLE 1 Percent Time to Time Assisted Unassisted Survival stand Feversto eat drinking Drinking (72 hrs (hrs) (%) (days) (days) (days) Treated100 4.21 57.14 1.24 1.56 3.13 Control 62.5 6.28 75.00 1.36 0.96 3.12

As seen in Table 2, gait analysis indicates improved motor function inNPEX treated pigs compared to controls. At 7 days post-stroke, NPEXtreated pigs move faster and with more cadence (rhythmicity) as theymove throughout their stride. Due to stroke in the right hemisphere, theleft side specific deficits were more pronounced. Left side specificmeasurements indicate greater step length, shorter cycle time, greaterstride length, and swing percent of cycle time. Treated animals placedmore pressure on each foot as they moved through their stride anddisplayed more pronounced movement of the hind limbs past the front limbevidenced by the reach, indicating a more natural movement compared tocontrols.

TABLE 2 Step Length Cycle Time Stride Length Swing % Foot Hind VelocityCadence (cm) (sec) (cm) of Cycle Pressure Reach NPEX 131.45 91.99 41.430.79 80.89 41.06 62.87 −12.34 Control 106.63 79.99 38.60 0.83 77.1438.28 57.18  −9.55

Results

Initial MRI analysis at 28±4 hours indicated a smaller lesion volume inNPEX treated pigs compared to controls (FIG. 10A-10C). Volumemeasurements of the ipsilateral and contralateral hemispheres indicateless change in volume after stroke with NPEX treatment (FIG. 10D),indicating less swelling in the ipsilateral hemisphere in the first 24hours after stroke, consistent with the rodent data indicating an earlymodulation of the immune response and indicative of a neuroprotectiveeffect in the porcine model as well. Physiological parameters, mostnotably survival, was also increased in the 72 hours following inductionof stroke. Treated animals were also able to stand unassisted ˜2 hoursfaster than controls. Gait analysis at 7 days post-stroke indicatedimproved motor function as detected by increased velocity and cadence(rhythmicity), more pressure being applied to each foot throughout themotion, as well as increased step and stride length, an increasedpercentage of time in swing stance (foot off of ground) per cycle, and amore pronounced reach with the rear limb extending past the fore-limb,as is expected in quadrupeds.

NPEX treatment also had a profound effect on molecular benefit in theanimals 12 weeks post-stroke as detected by MRI T2 and T2 FLAIR images(FIG. 11). Dead/dying tissue was reduced by treatment and damaged tissue(green traces) were far reduced, involving mainly cortical tissue whilelargely preserving integrity of the ventricle compared to the control.Animals that received NPEX were able to survive larger infarct sizes (asmuch as 3.8 cm3 larger at 24 hours post-stroke) over the 12 week studythan those that received control treatment. This is probably due to theanti-inflammatory properties of NPEX leading to decreased strokeseverity and promoting better long-term outcomes including integrity ofbrain tissue, as well as behavioral and motor function.

Never before have EVs of any kind improved the outcome of stroke or forthat matter any neural deficit in a large animal. Consistent with thesmall animal we show that NPEX effects the brain immediately, thus NPEXis fast acting in the pig and the mouse. We do not know of any otherstudy that has suggested such and immediate effect of EVs on a strokedanimal. Immediately NPEX improved survival and motor function (speed inwhich animals recover, time to stand, balance, etc). Molecular andphenotypic outcomes in a large animal species expand upon previouslydescribed rodent data, indicating that EVs exert a likelyneuroprotective effect that is longer lasting. This effect is likely duein part to modulation of the secondary injury cascade by muting theimmune response that occurs following stroke, as molecular andphenotypic differences are detectable as early as 24 hours post-strokeand a later effect on the enhanced M2 response, which isneuro-reparative. The longer lasting effects are observed throughout,with improved gait of the NPEX treated animals.

In summary, this is the first stroke study to suggest to mechanisms ofaction are consistent across animals and includes a study in a morecomplex brain with a structure that is similar to humans. Our in vitrohuman cell studies suggest neuroprotective action directly on thedamaged neural cells, and the mouse and pig studies suggest protectionthrough the immune system. The longer acting effects on neuro-reparativemechanisms in both species were observed and could be in part due to upregulation of Treg cells (mouse only to date). Importantly, the porcinebrain unlike the mouse shares many homologies with the human brainincluding white matter ratio, presence of gyrencephalic cortex,cytoarchitecture, and size. Due to these similarities, the pig isconsidered a superior model system compared to rodents, and is likelymore representative of anticipated benefits in human treatment ofischemic stroke with therapeutically produced neural progenitor derivedEVs.

This is possible due to an ability to produce a large quantity of EVsfrom neural progenitor cultures, approximately 5 times greater thanyields obtained from MSCs. The ability to generate EVs on a commercialscale consistently from cultures will require that EV-producing cells bedependent on a rigorous quality control process for handling the cellsand purifying the resultant EVs.

Example 6

Primary Filtration (Step 1, Used for all Purification Methods)

Media is harvested from plates or flasks containing cultured cells. Theharvested media was frozen at −20° C. before or after the primaryfiltration and thawed at 4° C. Filtration is completed in a sterilelaminar flow hood to minimize contamination. The harvested media wasfiltered via dead end filtration through a 0.22 μm filter (EMD MilliporeStericup). This primary filtration removes any cellular debris and/ordead cells from the harvested media. This allows for the EVs andmicrovescicles to pass through to the filtrate for further purification.

Centricon Ultrafiltration

Having undergone primary filtration, the media is ready for secondaryfiltration. This step utilizes a system including Centricon centrifugeunits, which can process up to 70 ml culture medium per spin. Thisfiltration process uses a 100 kDa filter to collect enriched EVs. Duringthis process the media is forced through the filter discs viacentrifugation at 4,000×g. Once the retentate reaches approximately 250μL to 5 mL per unit, a buffer exchange is then performed usingapproximately 90% of the starting media volume of PBS+/+. The PBS+/+ ispassed through the Centricon filters by centrifugation at 2,000×g untilthe retentate reaches 250 μL to 1 mL of volume per filter unit. At thispoint the retentate is collected for EV analysis. A sample is taken forimmediate nanoparticle tracking analysis, and purified EVs from the samecell type were pooled, triturated, and aliquoted into DNase/RNase freetubes for storage at −20° C.

Amicon Stirred-Cell Ultrafiltration

For larger volume ultrafiltrations, Amicon stirred-cell ultrafiltrationunits were used, connected 3 units in tandem. Each base unit held 400 mlmedium, which was further expanded using a reservoir to increasethroughput. This step utilizes a system including a pressure vessel, andthree EMD Millipore Stirred Cell Units (Fisher Scientific, USA) workingin sequence. This step utilizes dead end filtration as well with mediapassing through a 100 kDa filter disc (Fisher Scientific, USA). Duringthis process the media is forced through the filter discs using 25 psiof positive pressure supplied by compressed nitrogen gas. Once theretentate reaches 50 ml/stirred cell unit the system is de-pressurized.A buffer exchange is then performed using 50% of the starting mediavolume of PBS+/+. The PBS+/+ is passed through the stirred cell filtersuntil the retentate reaches 50 mL of volume per filter unit. At thispoint the retentate is collected for EV analysis. A sample is taken forimmediate nanoparticle tracking analysis, and purified EVs from the samecell type were pooled, triturated, and aliquoted into DNase/RNase freetubes for storage at −20° C.

Fibroblast Growth Factor ELISA

To determine hFGF2 content, cryopreserved samples of NPEX™, APEX, andMSCEX EVs were thawed at 4° C. and lysed with an equal volume of EVlysis buffer to release exosomal content. The lysate was analyzed forhuman FGF2 using a commercially available human FGF2 ELISA assay kit(Thermo Scientific—Catalog number KHG0021) following manufacturersprotocol. FGF2 standards provided with the kit were used to generate astandard curve and quantitated FGF2 in the test sample. FGF2 wasdetected in NPEX™ sample at 490 pG/mL. No FGF2 could be detected eitherin APEX™ or in MSCEX™ samples.

Mass Spectroscopy

To compare proteins unique to neural EVs versus MSC derived EVs,purified EV proteins were subjected to mass spectroscopy byBioproximity, LLC.

Results

Ultracentrifugation and ultrafiltration methods were used as previouslydescribed. Large volume purifications utilized the Amicon stirred-cellsystem to purify over 24 liters of cell culture medium in a single workweek.

Fibroblast Growth Factor ELISA Results

The protein human Fibroblast Growth Factor 2 (hFGF2) is added to hNP1™culture media to maintain the proliferative state of hNP1™ cells. EVsproduced by hNP1™ cells (NPEX) cultured in hFGF2 supplemented media mayaccumulate/contain any of this hFGF2 protein as part of the exosomalprotein content. NPEX EVs, harvested from hNP1™ culture media collectedfrom live hNP1™ cell cultures were tested for the presence of hFGF2.These purified and cryopreserved NPEX™ EVs contained detectable levelsof hFGF2 after thaw. On the other hand, EVs from hAstroPro™ humanastrocytes (APEX), and from mesenchymal stem cells (MSCEX), where thecell culture media is not supplemented with hFGF2, did not contain anydetectable hFGF2. Taken together, these data indicate that proteinssupplemented in the medium are taken up by the cells and are present inthe enriched EV sample even in the absence of transfection or othertechniques to overexpress proteins in the cells.

Mass Spectroscopy

FIG. 12 is a Venn diagram showing the number of proteins unique to, andshared by NPEX, APEX, and MSCEX vesicles. A total of 2727 proteins wereshared by all of these vesicles (Table 3). APEX and MSCEX shared 2786proteins that were not in NPEX vesicles (Table 4). NPEX shared 467proteins only with MSCEX (Table 5) and 426 proteins only with APEXvesicles (Table 6). MSCEX had 536 unique proteins that were notidentified in APEX or NPEX vesicles (Table 7). APEX had 596 proteinsthat were not identified in NPEX or MSCEX vesicles (Table 8). NPEX had1653 proteins that were not identified in APEX or MSCEX vesicles (Table9).

TABLE 3 Protein codes found in NPEX, APEX, and MSCEX 1 A0A023UFG1 2A0A024QZ30 3 A0A024R0R4 4 A0A024R1Q5 5 A0A024R269 6 A0A024R294 7A0A024R2A8 8 A0A024R3H6 9 A0A024R3W5 10 A0A024R473 11 A0A024R4U7 12A0A024R663 13 A0A024R6C6 14 A0A024R7F1 15 A0A024R816 16 A0A024R900 17A0A024R972 18 A0A024R9G7 19 A0A024RA85 20 A0A024RAB6 21 A0A024RB27 22A0A024RB49 23 A0A024RBR1 24 A0A024RC42 25 A0A024RD01 26 A0A024RDU6 27A0A087WTT9 28 A0A087WTY7 29 A0A087WU72 30 A0A087WUP3 31 A0A087WUR9 32A0A087WUZ8 33 A0A087WV01 34 A0A087WV58 35 A0A087WVQ9 36 A0A087WVY5 37A0A087WX41 38 A0A087X1N7 39 A0A087X208 40 A0A087X270 41 A0A088AWP7 42A0A090N7U3 43 A0A090N8G0 44 A0A090N8I2 45 A0A090N8Z3 46 A0A096LNH2 47A0A096LP10 48 A0A096LPK6 49 A0A0A0MQZ3 50 A0A0A0MR11 51 A0A0A0MRA3 52A0A0A0MRA8 53 A0A0A0MT16 54 A0A0A7NZX2 55 A0A0B4J262 56 A0A0C4DG44 57A0A0C4DGP4 58 A0A0C4DGQ0 59 A0A0C4DGR9 60 A0A0C4DGV7 61 A0A0C4DH10 62A0A0C4DH26 63 A0A0C4DH32 64 A0A0C4DH71 65 A0A0D9SF05 66 A0A0D9SF53 67A0A0D9SFF0 68 A0A0E3SU01 69 A0A0F7TC28 70 A0A0G2JQ57 71 A0A0J9YWL9 72A0A0J9YY17 73 A0A0J9YY65 74 A0A0K0K1J1 75 A0A0K2FPC8 76 A0A0R4J2G5 77A0A0S2Z3C0 78 A0A0S2Z3H6 79 A0A0S2Z3N2 80 A0A0S2Z4F6 81 A0A0S2Z4K6 82A0A0S2Z4S4 83 A0A0U1RQF0 84 A0A0U1RQK4 85 A0A0U1RR05 86 A0A0U1RRH6 87A0A0U4BW16 88 A0A0U4DR30 89 A0A0U5Q0I5 90 A0A0X7YLB8 91 A0A0X9T0I7 92A0A0X9TDD0 93 A0A0X9UWM4 94 A0A109PSY4 95 A0A126GVT4 96 A0A126GVV9 97A0A126GVY4 98 A0A126GW97 99 A0A126LAV0 100 A0A126LAW8 101 A0A140VJG3 102A0A140VJM5 103 A0A140VJZ1 104 A0A140VJZ4 105 A0A140VK24 106 A0A142K0N8107 A0N7J6 108 A1A5C4 109 A1L0U7 110 A1YZK0 111 A2IPH5 112 A2J1M8 113A2MYD2 114 A2N0U1 115 A2RRE5 116 A2RTY3 117 A3KMF7 118 A3KMG4 119 A4D0R5120 A4D1F6 121 A4D263 122 A4D2F6 123 A4FU99 124 A5XEJ8 125 A6NCJ1 126A6NDG6 127 A6NHK2 128 A6NMH8 129 A8K1Z4 130 A8K2Q7 131 A8K2X9 132 A8K4L4133 A8K580 134 A8K5W7 135 A8K646 136 A8K674 137 A8K6Q8 138 A8K6R3 139A8K889 140 A8K941 141 A8K948 142 A8KA19 143 A8KAM8 144 A8KAQ0 145 A8MQ14146 A8MSG4 147 A8MUM1 148 A8MYJ1 149 B0AZV0 150 B0I1R4 151 B0I1R7 152B0I1R8 153 B0I1S0 154 B1AH62 155 B2R604 156 B2R6E3 157 B2R8C2 158 B2R8R5159 B2RAG9 160 B2RAK1 161 B2RD27 162 B2RDG9 163 B2RDV2 164 B2RP65 165B2ZZ86 166 B3KMD3 167 B3KMX8 168 B3KNH6 169 B3KNH8 170 B3KNJ3 171 B3KR88172 B3KS22 173 B3KS78 174 B3KT94 175 B3KUL2 176 B3KV04 177 B3KVE3 178B3KVJ2 179 B3KW39 180 B3KX05 181 B3KXG9 182 B4DE59 183 B4DEA8 184 B4DEN3185 B4DGW2 186 B4DGX3 187 B4DH09 188 B4DHQ3 189 B4DHR1 190 B4DHV2 191B4DI57 192 B4DJ98 193 B4DJM8 194 B4DK16 195 B4DK41 196 B4DKE0 197 B4DKG5198 B4DKL5 199 B4DL04 200 B4DL67 201 B4DLV7 202 B4DM31 203 B4DMC6 204B4DMD7 205 B4DMI9 206 B4DMK9 207 B4DMS8 208 B4DMY4 209 B4DN39 210 B4DN66211 B4DN96 212 B4DNS4 213 B4DPB7 214 B4DPF0 215 B4DPS3 216 B4DQF8 217B4DQJ9 218 B4DRF2 219 B4DRV4 220 B4DRZ1 221 B4DRZ5 222 B4DSD8 223 B4DSI2224 B4DSK2 225 B4DTD5 226 B4DTF5 227 B4DTI4 228 B4DTV0 229 B4DUV1 230B4DVQ8 231 B4DVY2 232 B4DXJ3 233 B4DXW6 234 B4DXX8 235 B4DYE2 236 B4DYM1237 B4DYQ3 238 B4DYV9 239 B4DZ96 240 B4DZD8 241 B4DZK5 242 B4DZL0 243B4DZX5 244 B4DZY9 245 B4E0C3 246 B4E116 247 B4E1Z8 248 B4E2C9 249 B4E310250 B4E358 251 B4E396 252 B4E3S1 253 B5BUB5 254 B5MC96 255 B7SXT3 256B7WPN9 257 B7Z1C7 258 B7Z1Y1 259 B7Z208 260 B7Z225 261 B7Z2P6 262 B7Z395263 B7Z3F9 264 B7Z3I3 265 B7Z425 266 B7Z4S3 267 B7Z5R5 268 B7Z5U1 269B7Z6G1 270 B7Z6T9 271 B7Z865 272 B7ZAS5 273 B7ZKX2 274 B7ZKY2 275 B7ZL41276 B7ZL68 277 B7ZMC8 278 B7ZVX0 279 C0JYZ2 280 C4P0A0 281 C8CHS3 282C9IYI4 283 C9IZK7 284 C9J0E9 285 C9J6F5 286 C9J8V2 287 C9J9C4 288 C9JBI4289 C9JG87 290 C9JKM5 291 C9JKM9 292 C9JSJ3 293 C9JX31 294 D3DPG0 295D3DQH8 296 D3DTX7 297 D3DUZ3 298 D3DV53 299 D3DWL0 300 D3DX49 301 D3VVE1302 D3VVP9 303 D4YW75 304 D6RCE4 305 D6RCP5 306 D6RCQ3 307 D6RGX4 308D6RHD5 309 D6RHJ5 310 D6W573 311 D9ZGF2 312 E2QRN8 313 E5RFF8 314 E5RG49315 E7EMW7 316 E7EQW5 317 E7ESB3 318 E7ETY2 319 E7EUH7 320 E7EX80 321E9PB28 322 E9PDD2 323 E9PFQ4 324 E9PJ15 325 E9PJG1 326 E9PKH2 327 E9PKQ0328 E9PS68 329 F1LLU8 330 F5GZB4 331 F5H7Y6 332 F6X3N5 333 F8VBW7 334F8VPD4 335 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A0A0A6YYF7 1218 A0A0C4DGN2 1219 A0A0G2JN42 1220A0A0K0K1H8 1221 A0A0S2Z3Y1 1222 A0A0U1RR22 1223 A0A0U1RRB6 1224A0A125U0V4 1225 A0A126LAY2 1226 A0A140VKC0 1227 A1L0W4 1228 A4D111 1229A4D275 1230 A4D2D2 1231 A6NE02 1232 A7E2D5 1233 A7MBN3 1234 A8K0D3 1235A8K2T0 1236 A8K7E4 1237 A8K7F6 1238 A8K8N7 1239 A8MQB8 1240 B1ALU3 1241B1AQP2 1242 B2RDN9 1243 B2RWP0 1244 B2RXH2 1245 B3KRN2 1246 B3KUD3 1247B3KW75 1248 B3KW81 1249 B3KXS7 1250 B3KYA4 1251 B3Y1X6 1252 B4DDF8 1253B4DEC7 1254 B4DER6 1255 B4DET5 1256 B4DH02 1257 B4DH43 1258 B4DHS0 1259B4DHZ6 1260 B4DKR1 1261 B4DM50 1262 B4DPF7 1263 B4DPR2 1264 B4DSH6 1265B4DTD3 1266 B4DUY3 1267 B4DWR5 1268 B4DYL8 1269 B4E0A4 1270 B4E123 1271B4E1L0 1272 B4E1N8 1273 B4E1Q0 1274 B4E2H9 1275 B4E2S2 1276 B4E3R1 1277B7Z4A4 1278 B7Z4R3 1279 B7Z4V1 1280 B7Z5C0 1281 B7Z5M1 1282 B7Z7Z9 1283B7Z829 1284 B7Z983 1285 B7ZA86 1286 B7ZLS9 1287 C0LLI5 1288 C8CH86 1289C9IZN7 1290 C9JBW9 1291 C9JSD3 1292 C9K0F9 1293 D6RD73 1294 D6RFI7 1295D6W603 1296 D6W636 1297 E3UVQ2 1298 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Q8N5R8 1467 Q8N9W8 1468Q8NAA4 1469 Q8NDQ8 1470 Q8NG06 1473 Q8TC36 1472 Q8TE47 1473 Q9NRG7 1474Q9NS39 1475 Q9NVW7 1476 Q9P0K7 1477 Q9P0U3 1478 Q9P1C0 1479 Q9UBG5 1480Q9UFC3 1481 Q9ULE4 1482 Q9ULV4 1483 Q9UMS0 1484 Q9Y5T4 1485 S6AWE3 1486U3KPS5 1487 V9GYJ9 1488 A0A024R2E1 1489 A0A024R2M2 1490 A0A087WZM7 1491A0A0A0MRE3 1492 A0A0A0MSB3 1493 A0A0A0MTR1 1494 A0A0F7G8J1 1495A0A0H4PJU9 1496 A0A0S2Z4Z5 1497 A0A125SXV9 1498 A0A126LB06 1499A0A126LB27 1500 A0N4Z6 1501 A8K2X4 1502 A8K3Q2 1503 A8K486 1504 A8K9U11505 A9Z1X1 1506 B1ANH7 1507 B2R950 1508 B2R9K6 1509 B2RBG2 1510 B2RCP41511 B3KTN4 1512 B3KVW6 1513 B4DH07 1514 B4DHB3 1515 B4DI28 1516 B4DIG01517 B4DJG5 1518 B4DMW8 1519 B4DMX4 1520 B4DNA0 1521 B4DTV9 1522 B4DW331523 B4E1P8 1524 B4E3Q1 1525 B7WNR0 1526 B7Z920 1527 B7ZAA5 1528 B7ZBP31529 C9J6G3 1530 C9JC84 1531 C9JI87 1532 E5RIL7 1533 E7EQM8 1534 E7EUJ71535 E9PK10 1536 E9PM35 1537 E9PMF0 1538 F8VS81 1539 F8VTR5 1540 F8W6961541 F8WEP4 1542 H0Y596 1543 H0YM50 1544 H3BRJ5 1545 H7BZ61 1546 H7C0E21547 H7C185 1548 H7C3P7 1549 H7C3S6 1550 I0J9E2 1551 J3KP97 1552 J3KSC11553 K7EKZ1 1554 K9MS24 1555 M0R0S9 1556 O60701 1557 O95613 1558 P063311559 P07737 1560 P08253 1561 P11021 1562 P25391 1563 P30041 1564 P312671565 P32238 1566 P46093 1567 P53618 1568 P55786 1569 P58397 1570 P601741571 P62304 1572 Q05DH4 1573 Q0IIN9 1574 Q1HW68 1575 Q49SQ1 1576 Q4LE451577 Q59FG0 1578 Q5H9N7 1579 Q5VW36 1580 Q6AZ94 1581 A0A024QZJ6 1582A0A024R1X2 1583 A0A024R2J4 1584 A0A024R4X7 1585 A0A024R524 1586A0A024R694 1587 A0A024R6R4 1588 A0A024R8F8 1589 A0A024RAR5 1590A0A024RC65 1591 A0A024RDJ4 1592 A0A024RDP2 1593 A0A024RDY0 1594A0A075B6U6 1595 A0A087WTA2 1596 A0A087WW31 1597 A0A087WXI0 1598A0A087WXI2 1599 A0A087WY71 1600 A0A087WYL6 1601 A0A087WYS6 1602A0A087WZE5 1603 A0A087X115 1604 A0A087X1P2 1605 A0A087X2A5 1606A0A096LP50 1607 A0A096LPG1 1608 A0A0A0MSK6 1609 A0A0A0MSW5 1610A0A0A0MT87 1611 A0A0A0MTS7 1612 A0A0A0N0L5 1613 A0A0A8WIM0 1614A0A0C4DG40 1615 A0A0C4DGG6 1616 A0A0C4ZLG8 1617 A0A0D9SF38 1618A0A0E3DD76 1619 A0A0G2JH42 1620 A0A0G2JKA5 1621 A0A0G2JS76 1622A0A0H4IV28 1623 A0A0S2Z3F5 1624 A0A0S2Z421 1625 A0A0S2Z4Q2 1626A0A0S2Z4Y8 1627 A0A0S2Z500 1628 A0A0S2Z592 1629 A0A0U1RQE8 1630A0A0U1RR70 1631 A0A126GW51 1632 A0A126LAX2 1633 A0A126LB21 1634A0A126LB25 1635 A0A126LB36 1636 A0A140VK10 1637 A0A140VK26 1638A0A140VK66 1639 A0A140VKE5 1640 A0A158RFU5 1641 A0JLU2 1642 A0PJZ3 1643A2IPH7 1644 A2JA19 1645 A2V663 1646 A4D1E4 1647 A4D233 1648 A5PKX5 1649A6H8X0 1650 A6N865 1651 A6NCN8 1652 A6NCT7 1653 A6NEC2 1654 A6NFI3 1655A6NHR9 1656 A6NL46 1657 A6NLG9 1658 A6NMY6 1659 A6NMZ7 1660 A6PVY3 1661A7E2F9 1662 A8K2P8 1663 A8K343 1664 A8K394 1665 A8K468 1666 A8K4W7 1667A8K6K9 1668 A8K6T3 1669 A8K9T5 1670 A8KA30 1671 A8KAJ9 1672 A8MZI9 1673B0AZV7 1674 B0LPE5 1675 B1AML2 1676 B1N7G3 1677 B1WB49 1678 B2R4Q7 1679B2R5M9 1680 B2R6N0 1681 B2R6N6 1682 B2R7N9 1683 B2R926 1684 B2RAH7 1685B2RB89 1686 B2RBF5 1687 B2RD36 1688 B2RD79 1689 B2RDX5 1690 B2RG98 1691B3CJD3 1692 B3KNV5 1693 B3KP07 1694 B3KPN7 1695 B3KQG6 1696 B3KQK3 1697B3KQT2 1698 B3KRN6 1699 B3KRS1 1700 B3KSV8 1701 B3KTA3 1702 B3KTH9 1703B3KTQ9 1704 B3KU00 1705 B3KUB1 1706 B3KUX9 1707 B3KWK3 1708 B3KXJ7 1709B3KYA0 1710 B3W6G7 1711 B4DDK9 1712 B4DDQ4 1713 B4DG76 1714 B4DGF9 1715B4DI39 1716 B4DIQ8 1717 B4DJD3 1718 B4DKU3 1719 B4DM05 1720 B4DMF0 1721B4DNE9 1722 B4DNI5 1723 B4DPM5 1724 B4DPV4 1725 B4DR01 1726 B4DR16 1727B4DR46 1728 B4DSR6 1729 B4DSU7 1730 B4DT39 1731 B4DU16 1732 B4DUD7 1733B4DUZ6 1734 B4DVQ0 1735 B4DW60 1736 B4DXM0 1737 B4DYJ4 1738 B4DZ36 1739B4DZP6 1740 B4DZQ7 1741 B4E1P9 1742 B4E3R4 1743 B5MCY1 1744 B5MD39 1745B5MEF5 1746 B6ZGT4 1747 B7Z2B7 1748 B7Z3I1 1749 B7Z3R6 1750 B7Z3V1 1751B7Z4S2 1752 B7Z5H8 1753 B7Z6I6 1754 B7Z6K5 1755 B7Z704 1756 B7Z7Z0 1757B7Z938 1758 B7Z9B7 1759 B7Z9J7 1760 B7Z9W3 1761 B7ZKM7 1762 B7ZLE5 1763C1K3N0 1764 C6KIA6 1765 C9JPF8 1766 C9JQ82 1767 C9JQI7 1768 C9JSP6 1769C9JTC5 1770 C9JTN7 1771 C9JY46 1772 D2X9V0 1773 D3DN94 1774 D3DPF9 1775D3DR43 1776 D3DU04 1777 D3DVC4 1778 D3DW55 1779 D3DX93 1780 D3VVD3 1781D3WYY9 1782 D6R9T4 1783 D6REL0 1784 D6RIE4 1785 D6RJB7 1786 D6W5C2 1787D7RTA7 1788 E5RFV7 1789 E5RHN3 1790 E5RIY1 1791 E5RJA3 1792 E7ENR6 1793E7EQ34 1794 E7ER60 1795 E7ERF0 1796 E7ESF5 1797 E7EWS1 1798 E9PAL5 1799E9PBB1 1800 E9PC41 1801 E9PDN5 1802 E9PII3 1803 E9PJ29 1804 E9PKB8 1805E9PKI9 1806 E9PLK6 1807 E9PM30 1808 E9PMQ3 1809 E9PMV1 1810 E9PNF1 1811E9PNZ6 1812 E9PPM7 1813 E9PQ56 1814 E9PQL1 1815 E9PRA7 1816 F4ZW64 1817F5GXA0 1818 F5GY56 1819 F5H423 1820 F5H7E5 1821 F5H8I3 1822 F8VSC5 1823F8VUZ7 1824 F8VWV6 1825 F8W1S1 1826 F8WAM2 1827 F8WBI5 1828 F8WBJ2 1829FSWCF2 1830 F8WD09 1831 F8WDF1 1832 F8WDM8 1833 F8WEK9 1834 G3V1N2 1835G3V2D8 1836 G3V5L2 1837 H0Y3T6 1838 H0Y592 1839 H0Y938 1840 H0Y9K1 1841H0YA55 1842 H0YAD3 1843 H0YAM0 1844 H0YAS1 1845 H0YBNS 1846 H0YCL7 1847H0YCP4 1848 H0YEX9 1849 H0YFA9 1850 H0YFT1 1851 H0YIA0 1852 H0YIC5 1853H0YJP0 1854 H0YJV3 1855 H0YL56 1856 H0YM11 1857 H0YNY7 1858 H3BM10 1859H3BNR2 1860 H3BPD2 1861 H3BTE9 1862 H6VRF9 1863 H7BXN1 1864 H7BYJ3 1865H7BZ08 1866 H7BZI1 1867 H7C0D8 1868 H7C0Y8 1869 H7C0Z9 1870 H7C269 1871H7C435 1872 H7C4A7 1873 H7C4G9 1874 H7C505 1875 H7C519 1876 H7C5R4 1877H9MHK7 1878 I0CMK6 1879 I1SRC5 1880 I3L192 1881 I3L202 1882 I3L2B0 1883I3L454 1884 I6U4P1 1885 J3KNM9 1886 J3KRI7 1887 J3KRQ5 1888 J3KRS3 1889J3KRZ0 1890 J3KSI7 1891 J3QKM3 1892 J3QR31 1893 J3QR90 1894 K7EKP8 1895K7EQJ8 1896 K7ES02 1897 K7ES57 1898 L0B1V4 1899 L0R8A8 1900 L7RXH0 1901L8E775 1902 L8E7T8 1903 L8E872 1904 L8E935 1905 L8EA23 1906 L8EC78 1907L8ECD0 1908 L8ECG5 1909 L8ECJ9 1910 L8ECM9 1911 M0QX38 1912 M0QZ52 1913M0QZH0 1914 M0QZL2 1915 M0R0B6 1916 M0R0I5 1917 M0R1Y0 1918 M0R265 1919M0R2B8 1920 M0R2G8 1921 M0R2X8 1922 O00186 1923 O00228 1924 O00231 1925O00300 1926 O00370 1927 O00602 1928 O15031 1929 O15061 1930 O15379 1931O15389 1932 O15400 1933 O43298 1934 O43313 1935 O60318 1936 O60687 1937O60806 1938 O60869 1939 O60888 1940 O75037 1941 O75051 1942 O75054 1943O75230 1944 O75294 1945 O77727 1946 O94933 1947 O95073 1948 O95396 1949O95459 1950 O95622 1951 O95760 1952 P00761 1953 P01024 1954 P01031 1955P01716 1956 P01782 1957 P04264 1958 P05388 1959 P07996 1960 P08572 1961P09544 1962 P09619 1963 P0C091 1964 P0C671 1965 P0C7M7 1966 P0C7V6 1967P0CG21 1968 P0DKB5 1969 P10745 1970 P12109 1971 P12268 1972 P13196 1973P15531 1974 P15621 1975 P19174 1976 P20783 1977 P22760 1978 P23471 1979P25786 1980 P25788 1981 P25940 1982 P27694 1983 P30050 1984 P30101 1985P31152 1986 P33908 1987 P35555 1988 P35606 1989 P35908 1990 P36955 1991P38570 1992 P38606 1993 P38935 1994 P45974 1995 P47989 1996 P49419 1997P49454 1998 P49588 1999 P49788 2000 P49792 2001 P50570 2002 P51160 2003P51884 2004 P53708 2005 P54136 2006 P56537 2007 P61221 2008 P62318 2009P62805 2010 P69905 2011 P82987 2012 P98160 2013 Q01959 2014 Q02388 2015Q02779 2016 Q04721 2017 Q05BT5 2018 Q05BU1 2019 Q05BV1 2020 Q05C82 2021Q05CV2 2022 Q06210 2023 Q07954 2024 Q08211 2025 Q0VGA3 2026 Q0ZAJ2 2027Q0ZAJ7 2028 Q12813 2029 Q12965 2030 Q13118 2031 Q13136 2032 Q13162 2033Q13537 2034 Q14127 2035 Q14315 2036 Q14423 2037 Q14543 2038 Q14917 2039Q14C87 2040 Q14CE9 2041 Q15058 2042 Q15113 2043 Q15247 2044 Q15296 2045Q16394 2046 Q16761 2047 Q2I009 2048 Q2M1Z3 2049 Q2M2I5 2050 Q2M3M2 2051Q2PT48 2052 Q2WEN9 2053 Q2WGN9 2054 Q3T906 2055 Q494V2 2056 Q494X3 2057Q496A9 2058 Q496Y1 2059 Q499G7 2060 Q4KMG9 2061 Q4LE48 2062 Q4VBP4 2063Q53H82 2064 Q53HC2 2065 Q53HI2 2066 Q53HQ4 2067 Q53HU6 2068 Q562R1 2069Q562V5 2070 Q562Z4 2071 Q59E18 2072 Q59FG2 2073 Q59FM3 2074 Q59FS8 2075Q59GB4 2076 Q59GM9 2077 Q59GN1 2078 Q59H94 2079 Q59HE3 2080 Q5CZB5 2081Q5F1R6 2082 Q5H9M0 2083 Q5H9S0 2084 Q5HYC2 2085 Q5J875 2086 Q5JPB2 2087Q5JXI3 2088 Q5JY65 2089 Q5JZ02 2090 Q5K684 2091 Q5R2U7 2092 Q5RI53 2093Q5SQS7 2094 Q5SR54 2095 Q5T6X5 2096 Q5T7P6 2097 Q5T8A7 2098 Q5TCT4 2099Q5TFJ7 2100 Q5U0K8 2101 Q5VWW1 2102 Q5VZB9 2103 Q5XKR4 2104 Q63HL4 2105Q641R5 2106 Q659F9 2107 Q68CZ6 2108 Q68D85 2109 Q68VJ5 2110 Q6EMK4 2111Q6FGB3 2112 Q6IBG1 2113 Q6IBZ4 2114 Q6IPQ0 2115 Q6MZX7 2116 Q6NVY8 2117Q6NXN2 2118 Q6NXP6 2119 Q6P2D8 2120 Q6P461 2121 Q6PGP7 2122 Q6TFL4 2123Q6U717 2124 Q6UVK1 2125 Q6UW63 2126 Q6UWF9 2127 Q6UWP8 2128 Q6UWX4 2129Q6DXR4 2130 Q6UXS9 2131 Q6Y2K9 2132 Q6ZNE2 2133 Q6ZP05 2134 Q6ZQP0 2135Q6ZTY9 2136 Q6ZUX1 2137 Q6ZUZ3 2138 Q70Z35 2139 Q71V99 2140 Q75MY8 2141Q75N88 2142 Q7RTY8 2143 Q7Z3A2 2144 Q7Z4Q2 2145 Q7Z5P9 2146 Q7Z5Y4 2147Q86VH2 2148 Q86W28 2149 Q86X55 2150 Q86XX4 2151 Q8IX54 2152 Q8IY85 2153Q8IYV9 2154 Q8IYX0 2155 Q8IZ13 2156 Q8N0Y7 2157 Q8N2E1 2158 Q8N3G4 2159Q8N5C1 2160 Q8N609 2161 Q8N7E2 2162 Q8N7U5 2163 Q8N944 2164 Q8N9V7 2165Q8NB66 2166 Q8ND82 2167 Q8NEN9 2168 Q8NFX7 2169 Q8NHP8 2170 Q8TBP0 2171Q8TD16 2172 Q8TD90 2173 Q8TDV0 2174 Q8TEY7 2175 Q8WUG5 2176 Q8WUH2 2177Q8WUJ3 2178 Q8WUM6 2179 Q8WWU8 2180 Q8WXI7 2181 Q8WY44 2182 Q8WZ18 2183Q8WZ31 2184 Q8WZB3 2185 Q92626 2186 Q92630 2187 Q92743 2188 Q92833 2189Q969Y0 2190 Q96AW0 2191 Q96BN2 2192 Q96BY6 2193 Q96DJ8 2194 Q96FN9 2195Q96HJ3 2196 Q96IT5 2197 Q96JQ0 2198 Q96KQ4 2199 Q96L96 2200 Q96LM5 2201Q96LQ0 2202 Q96MK8 2203 Q96MX6 2204 Q96N75 2205 Q96PD2 2206 Q96PD5 2207Q96PQ9 2208 Q96Q15 2209 Q96QH0 2210 Q96RS6 2211 Q96RW7 2212 Q99985 2213Q9BQK8 2214 Q9BRH9 2215 Q9BS26 2216 Q9BUG9 2217 Q9BWV1 2218 Q9BXJ3 2219Q9BXX0 2220 Q9BY89 2221 Q9BYX7 2222 Q9BZT5 2223 Q9C055 2224 Q9C071 2225Q9C0G0 2226 Q9CSP3 2227 Q9H1B7 2228 Q9H224 2229 Q9H2M8 2230 Q9H3U1 2231Q9H3U2 2232 Q9H526 2233 Q9H557 2234 Q9H7T9 2235 Q9H7U1 2236 Q9H814 2237Q9H863 2238 Q9H993 2239 Q9H996 2240 Q9HAR7 2241 Q9HBV4 2242 Q9HBW1 2243Q9HC10 2244 Q9HD43 2245 Q9NQL0 2246 Q9NQX0 2247 Q9NRN1 2248 Q9NS98 2249Q9NSF5 2250 Q9NT48 2251 Q9NU22 2252 Q9NUL7 2253 Q9NVD3 2254 Q9NXD9 2255Q9NYQ8 2256 Q9NYU2 2257 Q9NZ56 2258 Q9NZI8 2259 Q9NZJ4 2260 Q9NZQ3 2261Q9P0K0 2262 Q9P0Z3 2263 Q9P104 2264 Q9P186 2265 Q9P1F0 2266 Q9P266 2267Q9P2C8 2268 Q9P2R9 2269 Q9UBK8 2270 Q9UBW8 2271 Q9UCR1 2272 Q9UE50 2273Q9UF29 2274 Q9UGK2 2275 Q9UIY3 2276 Q9UJX5 2277 Q9UKE0 2278 Q9UKX2 2279Q9UL97 2280 Q9ULH4 2281 Q9ULI3 2282 Q9ULL8 2283 Q9UMG4 2284 Q9UMJ0 2285Q9UML5 2286 Q9UMR3 2287 Q9UN70 2288 Q9UPX8 2289 Q9UPY3 2290 Q9UPZ3 2291Q9UQR0 2292 Q9Y277 2293 Q9Y2K3 2294 Q9Y3A2 2295 Q9Y3D6 2296 Q9Y3I0 2297Q9Y4K0 2298 Q9Y574 2299 Q9Y5G4 2300 Q9Y5Q9 2301 Q9Y680 2302 Q9Y6C2 2303R4GMQ5 2304 R4GN15 2305 S4R3C7 2306 S4R3Q3 2307 U3KQG5 2308 U5IRJ4 2309V9GY54 2310 V9GZ55 2311 V9GZG2 2312 V9H0A6 2313 V9H102 2314 V9HW11 2315V9HW34 2316 V9HW62 2317 V9HW88 2318 V9HWB4 2319 V9HWB5 2320 V9HWE1 2321V9HWF2 2322 V9HWI6 2323 V9TNI8 2324 W8YM35 2325 X5DNI9 2326 X5DP31 2327X5DPA6 2328 X5DR62 2329 X6R647 2330 X6RE28 2331 A0A024R3N3 2332A0A024R456 2333 A0A024R462 2334 A0A024R6P6 2335 A0A024R7W9 2336A0A024R893 2337 A0A024R9I0 2338 A0A024R9Q1 2339 A0A024RAA7 2340A0A024RC00 2341 A0A087WTA8 2342 A0A087WUA7 2343 A0A087WVP1 2344A0A087WYF9 2345 A0A087X163 2346 A0A087X1V8 2347 A0A0A0MRD2 2348A0A0A0MRS2 2349 A0A0A0MSD0 2350 A0A0C4DFM7 2351 A0A0C4DG73 2352A0A0D9SFA9 2353 A0A0E3DCV7 2354 A0A0J9YVW2 2355 A0A0J9YVW5 2356A0A0J9YX90 2357 A0A0S2Z4B5 2358 A0A0X9UWJ6 2359 A0A126GVE6 2360A0A126LB13 2361 A1L4H1 2362 A6NEQ2 2363 A6PVK5 2364 A8K0K1 2365 A8K0P82366 A8K0R7 2367 A8K2P0 2368 A8K7T4 2369 A8K874 2370 A8K8X0 2371 A8K9642372 A8MTJ3 2373 B0AZS6 2374 B3KM36 2375 B3KNK5 2376 B3KPZ8 2377 B3KQU22378 B3KWQ8 2379 B3KX74 2380 B4DEB0 2381 B4DEG1 2382 B4DFY0 2383 B4DGQ72384 B4DGQ8 2385 B4DIP4 2386 B4DJ30 2387 B4DL30 2388 B4DM84 2389 B4DMV82390 B4DP50 2391 B4DPV2 2392 B4DPY2 2393 B4DRQ4 2394 B4DU77 2395 B4DW522396 B4E0D8 2397 B4E2L2 2398 B4E2M7 2399 B4E2Y4 2400 B7Z1H7 2401 B7Z2V62402 B7Z4C0 2403 B7Z6W4 2404 B7ZLW8 2405 B7ZMN7 2406 C6GLW5 2407 C9JH442408 C9JR58 2409 C9JW04 2410 D6R9I8 2411 D6REZ4 2412 E1A689 2413 E5RJI52414 E9PAV3 2415 E9PL83 2416 E9PPG5 2417 F1T0E5 2418 F2Z2U4 2419 F6QYI92420 F8W0W6 2421 F8W8H5 2422 G3V471 2423 H0Y645 2424 H0YAB8 2425 H0YB132426 H0YJ11 2427 H3BRX4 2428 H3BT74 2429 B3BUH4 2430 H7BZH1 2431 H7C1492432 H7C2W8 2433 I6L894 2434 I6L957 2435 J3KQU2 2436 J3QLS9 2437 K0P7932438 K4DIA0 2439 K4K7V6 2440 K7EQ63 2441 L0CQ38 2442 M0QZR9 2443 M0R2612444 M0R315 2445 O00420 2446 O14980 2447 O15240 2448 O43854 2449 O756912450 O95243 2451 P00800 2452 P01833 2453 P05109 2454 P0C869 2455 P0CB472456 P0CG39 2457 P0DKL9 2458 P12757 2459 P26022 2460 P28070 2461 P354422462 P35558 2463 P43652 2464 P54753 2465 P55089 2466 Q05CW1 2467 Q090282468 Q0VAS5 2469 Q14455 2470 Q16281 2471 Q32MQ0 2472 Q53HB3 2473 Q53TR02474 Q59EG0 2475 Q59F22 2476 Q59FG6 2477 Q59H91 2478 Q5BKT1 2479 Q5FBY72480 Q5T1R4 2481 Q5TBK7 2482 Q5TH58 2483 Q658L2 2484 Q658N2 2485 Q684P52486 Q69YM0 2487 Q6IC83 2488 Q6L9N1 2489 Q6N097 2490 Q6NUJ9 2491 Q6NUN22492 Q6NUP7 2493 Q6NXR7 2494 Q6P0Q1 2495 Q6P2I0 2496 Q6P5R7 2497 Q6PJ722498 Q6PK65 2499 Q6QE20 2500 Q6TCJ2 2501 Q6TFL3 2502 A0A075B6H0 2503A0A0A0MSY2 2504 A0A0B4J1Z7 2505 A0A0B4J2E5 2506 A0A0J9YXN7 2507A0A0S2Z4J7 2508 A4D2H0 2509 A5PLN9 2510 A8K2M0 2511 A8K7H1 2512 A8K9P02513 B2R769 2514 B2RA94 2515 B3KMG3 2516 B3KNF6 2517 B3KSB9 2518 B3KSG02519 B3KSJ0 2520 B3KU66 2521 B4DJB9 2522 B4DN12 2523 B4DNP9 2524 B4DPX62525 B4DQ18 2526 B4DQL5 2527 B4DTK1 2528 B7Z1Z9 2529 B7Z5P0 2530 C7TPG72531 C9J5J4 2532 C9JYJ6 2533 D3DPB7 2534 D6RHV3 2535 D7RF68 2536 E0CX092537 E1U340 2538 E5RH59 2539 E5RJV2 2540 E7EPF0 2541 E7ESP4 2542 E7EW932543 E9PQ73 2544 F5GX09 2545 F5GYN0 2546 F5H4E5 2547 F5H6Q4 2548 F8VWA32549 F8W1K8 2550 G3V599 2551 H0UI12 2552 H0Y853 2553 H0YAH0 2554 H0YFK02555 H3BPJ7 2556 H3BPS9 2557 H3BTH8 2558 H7C0I7 2559 H7C4L7 2560 J3KS132561 J3QS03 2562 L0R5C4 2563 O75603 2564 P08588 2565 P0CE72 2566 P207422567 P21453 2568 P35251 2569 P49720 2570 Q14766 2571 Q15333 2572 Q153892573 Q15631 2574 Q17RW2 2575 Q53RS3 2576 Q53S27 2577 Q53T09 2578 Q5BN462579 Q5RHP9 2580 Q5T6X2 2581 Q5TCI4 2582 Q5VX52 2583 Q6AHZ7 2584 Q6IBJ02585 Q6NWP9 2586 Q6P5T1 2587 Q6PIP7 2588 Q6U2F8 2589 Q6UXN8 2590 Q6UXQ02591 Q6Y288 2592 Q6ZMS2 2593 Q6ZMU0 2594 Q6ZMY6 2595 Q6ZNI2 2596 Q6ZNS12597 Q6ZQQ2 2598 Q6ZQY1 2599 Q6ZRK5 2600 Q6ZRK6 2601 Q6ZRM8 2602 Q6ZU102603 Q6ZV46 2604 Q6ZWC0 2605 Q6ZWE2 2606 Q6ZWG9 2607 Q70JA7 2608 Q71F562609 Q71U70 2610 Q75RY1 2611 Q765P7 2612 Q7LGC8 2613 Q7Z418 2614 Q7Z5272615 Q7Z528 2616 Q7Z5A3 2617 Q7Z5L7 2618 Q7Z5P4 2619 Q7Z5W6 2620 Q7Z5Y72621 Q7Z7L8 2622 Q7Z7M0 2623 Q7Z7M9 2624 Q86SQ6 2625 Q86SV6 2626 Q86TJ92627 Q86UD1 2628 Q86V85 2629 Q86VD1 2630 Q86VY4 2631 Q86WI1 2632 Q86XP02633 Q86YP6 2634 Q8IUZ8 2635 Q8IV28 2636 Q8IV92 2637 QSIVE3 2638 Q8IWD52639 Q8IXM7 2640 Q8IY33 2641 Q8IYA7 2642 Q8IYF3 2643 Q8IYP2 2644 Q8IZ522645 Q8IZD9 2646 Q8IZK6 2647 Q8IZQ8 2648 Q8N1G2 2649 Q8N2C7 2650 Q8N3972651 Q8N4T4 2652 Q8N715 2653 Q8N7P7 2654 Q8N7S3 2655 Q8N8V4 2656 Q8N9042657 Q8N987 2658 Q8N9V6 2659 Q8NA33 2660 Q8NAV8 2661 Q8NAY8 2662 Q8NB822663 Q8NCD8 2664 Q8ND61 2665 Q8NE22 2666 Q8NEZ4 2667 Q8TA93 2668 Q8TAS62669 Q8TB82 2670 Q8TD20 2671 Q8TDY8 2672 Q8TE73 2673 Q8WUV3 2674 Q8WXC62675 Q8WXE0 2676 Q92861 2677 Q969F2 2678 Q96AA8 2679 Q96BY7 2680 Q96JF02681 Q96KN7 2682 Q96KP6 2683 Q96M27 2684 Q96M95 2685 Q96N28 2686 Q96QE32687 Q96QZ0 2688 Q96S01 2689 Q96T80 2690 Q96TB4 2691 Q99715 2692 Q9BWG12693 Q9C005 2694 Q9H1K6 2695 Q9H212 2696 Q9H382 2697 Q9H3R1 2698 Q9H4L72699 Q9HBR0 2700 Q9HBU3 2701 Q9HD29 2702 Q9NQ33 2703 Q9NQW1 2704 Q9NR482705 Q9NTB9 2706 Q9NZ53 2707 Q9P2B2 2708 Q9UC91 2709 Q9UDL0 2710 Q9UHK02711 Q9UL81 2712 Q9ULI1 2713 Q9ULL0 2714 Q9ULL1 2715 Q9ULM8 2716 Q9UPU52717 Q9UPU9 2718 Q9Y3Q7 2719 S4R3E2 2720 U5XN63 2721 U6FSN9 2722 V9GYK62723 V9H019 2724 V9HW33 2725 V9HW38 2726 V9HW43 2727 X2L7S8

TABLE 4 Proteins in both MSCEX and APEX 1 A0A024R0P8 2 A0A024R3G0 3A0A087WVV2 4 A0A087WXZ2 5 A0A0A0MR07 6 A0A0A0MSZ4 7 A0A0G2JIC2 8A0A0K0K1H9 9 A0A0S2Z4G9 10 A0A140VK05 11 A0A140VK46 12 A2RU30 13 A2VCQ414 A4PB67 15 A8K3Y6 16 A8KAQ8 17 A8MXT8 18 B1AKG0 19 B2R7P8 20 B2R8Z8 21B2RBJ8 22 B3KMD9 23 B3KQF9 24 B3KQX9 25 B3KVF9 26 B4DDT3 27 B4DE33 28B4DFP1 29 B4DHX7 30 A0A024RA75 31 A0A024RDS3 32 A0A087WV40 33 A0A087WYY534 A0A0C4DFX3 35 A0A0J9YX34 36 A1JUI8 37 A1L4G8 38 A6NHB5 39 A7J1R1 40A8K2H9 41 A8K3B0 42 A8K4S1 43 A8K5U9 44 A8K7A4 45 B1AKQ8 46 B2R780 47B2RAQ9 48 B3KNK9 49 B3KVV5 50 B3KXA5 51 B4DH55 52 B4DKZ3 53 B4DL46 54B4DNX1 55 B4DQF6 56 B4DQQ9 57 B4DRV9 58 B4DS32 59 B4DUQ1 60 B4DUY7 61B4DVU1 62 B4DWG4 63 B4DWH5 64 B4DZN3 65 B4E047 66 B4E3M5 67 B7Z1I2 68B7Z2W3 69 B7Z2Z8 70 B7Z6U7 71 B7ZMB3 72 B8Y0L3 73 B8ZZ80 74 B9ZVT1 75C5HTY9 76 C9J406 77 C9J7H8 78 C9J9E8 79 C9JJV6 80 C9JNG9 81 C9JPM3 82D3DS95 83 A0A087X1Q2 84 A0A087X225 85 A0A0A0MTC7 86 A0A0S2Z430 87 A8K2T988 B4DE36 89 B4DMR3 90 B4E2A1 91 B4E3S2 92 B9EK61 93 C9JEL4 94 C9JXX4 95A0A087X0T3 96 A0A0S2Z3J5 97 B3KUJ8 98 B4DM82 99 B4DP93 100 B4DS46 101B4DY39 102 B7Z2Y4 103 C9J673 104 A0A024R8L6 105 A0A024RBZ8 106A0A087WUI7 107 A0A0B4J1Z4 108 A0A0S2Z3L7 109 A0A0S2Z4I7 110 A0A0U1RQJ2111 A0A0U1RQK7 112 A2A352 113 A0A024R1N1 114 A0A024R8F1 115 A0A087WVD1116 A0A0C4DGF5 117 A0A0C4DGS5 118 A0A0S2Z542 119 A0A140TA45 120A0A140VJW5 121 A4FVC0 122 A4QPE1 123 A5PKV2 124 A6NEM2 125 A8K5S1 126A8K6S1 127 A8K9T9 128 B1AMW7 129 B1Q3B3 130 B2R577 131 B2R7F8 132 B2R7M3133 B2R7T2 134 B2R8H4 135 B3KN59 136 B3KQT9 137 B3KS79 138 B3KSG1 139B3KSR8 140 B3KTM9 141 B3KTP2 142 B3KTP9 143 B3KW21 144 B3KWF2 145 B3KXY6146 B4DE00 147 B4DF60 148 B4DGH6 149 B4DH24 150 B4DI69 151 B4DID5 152B4DK14 153 B4DL98 154 B4DLA1 155 B4DLA3 156 B4DNR7 157 B4DPJ4 158 B4DRT0159 B4DTX0 160 B4E368 161 B7Z1D9 162 B7Z992 163 B8ZZF3 164 C9J4J8 165C9J4Z7 166 C9JBE1 167 C9JDW2 168 CQJJP8 169 CQJUM1 170 C9JZG1 171 C9JZW3172 C9K028 173 D3DQB3 174 D3DQI7 175 D3DWY7 176 D3DX70 177 D3K0R1 178D6RAL9 179 A0A024R0G8 180 A0A024R1Y8 181 A0A024RCZ4 182 A0A087WTQ6 183A0A087X2E9 184 A0A140VK29 185 B1ALD0 186 B2RBA0 187 B3KWJ0 188 B4DGD7189 B4DKI0 190 B4DM67 191 A6NE76 192 A6NJA2 193 A9LSU1 194 B4DDL1 195B4DIV8 196 B4DNH8 197 B4DP22 198 B4DR58 199 B4DTN6 200 B4DX99 201 B4DYQ7202 B4E0S6 203 B4E2M0 204 B7Z2S7 205 B7Z2V8 206 B7Z864 207 C9JPU9 208C9JZ53 209 D6R972 210 A0A024R0G0 211 A0A024R4C5 212 A0A024R7Z5 213A0A024RC72 214 A0A087WUW5 215 A0A087WVV1 216 A0A087X1N8 217 A0A087X232218 A0A0A0MT20 219 A0A0N7A6P0 220 A0A0U1RQV3 221 A0A140TA54 222 A0AV88223 A3KC71 224 A4QPB0 225 A7E2Y1 226 A7LFP5 227 A8K6I4 228 B1ALC0 229B2R514 230 B2R6L0 231 B2R7J8 232 B2RCM3 233 B2ZZ90 234 B3KM30 235 B3KMW3236 B3KP89 237 B3KR36 238 B3KUS2 239 B3KW84 240 B4DFR3 241 B4DG30 242A0A024R879 243 A0A087WYV9 244 A0A087X054 245 A0A087X0S5 246 A0A0D9SG88247 A0A0E3JSF5 248 A0A0S2Z3W2 249 A0A0S2Z6B4 250 A0A0U1RRC9 251 A0PJC9252 A1KY36 253 A4UCT2 254 A8K0G3 255 A8K709 256 A8K8Z1 257 A8KAY3 258B2R8G9 259 B2RBF8 260 B3KWG6 261 B3KY55 262 B4DHT1 263 B4DI54 264 B4DKW1265 B4DL92 266 B4DLZ2 267 B4DMF7 268 B4DPQ6 269 B4DPS0 270 B4DPU0 271B4DPU6 272 B4DQ56 273 B4E0N9 274 B4E261 275 B4E302 276 B7Z273 277 B7Z2M1278 B7Z4F6 279 B7Z4W2 280 B7Z856 281 B7Z9M4 282 B7ZAJ4 283 B7ZB44 284B7ZLI7 285 B7ZM04 286 B9DI82 287 B9EGI2 288 C0JYZ1 289 C1PHA2 290 C4P0D4291 C9J315 292 C9J9C1 293 C9JKA9 294 C9JN98 295 D3DPK5 296 D3DPU8 297D3DUK1 298 A0A024R1A3 299 A0A024RC58 300 A0A0S2Z5A5 301 A8K2R3 302A8K482 303 A8K6V0 304 B1ALD9 305 B1B5R8 306 B2RDG0 307 B3KU23 308 B3KUI4309 B4DGN8 310 B4DIW7 311 B4E3I6 312 C9J539 313 A0A087WX80 314A0A140VK07 315 B3KUJ2 316 B3KXN4 317 B4DJF2 318 B4DM34 319 A0A087WZ85320 B2RCP7 321 B3KNZ4 322 A0A024R127 323 A0A024R755 324 A0A024RDT5 325A0A087WUV5 326 A0A087WUZ3 327 A0A0A0MSX9 328 A0A0A7C7U2 329 A0A0C4DG95330 A0A0C4DH07 331 A0A0D9SG74 332 A0A0J9YVZ6 333 A0A0J9YWZ2 334A0A0S2Z3K0 335 A0A0U1RRA4 336 A0A140VJF9 337 A4UCT0 338 A8K7E0 339A8KA38 340 A8KA74 341 B1AH99 342 B2RCA1 343 B2RCJ7 344 B2RDI7 345 B3KN37346 B3KNG6 347 B3KNX0 348 B3KPI8 349 B3KQH1 350 B3KRC6 351 B3KRN4 352B3KS36 353 B3KS49 354 B3KSB2 355 B3KT90 356 B3KTR9 357 B3KUP0 358 B3KY30359 B4DDH3 360 B4DFM1 361 B4DL06 362 B4DM22 363 B4DM79 364 B4DQH6 365B4DUI5 366 B4E0H8 367 B7Z1H4 368 B7Z213 369 B7Z2N5 370 B7Z5S9 371 B7Z658372 B7Z7E5 373 B9EIS5 374 C9J1P7 375 C9JC48 376 C9JUP7 377 C9K031 378D6R9L2 379 D6RAF4 380 D6RAX7 381 D6RB21 382 D6RBE9 383 D6RBJ7 384 D6RCL6385 D6RDP1 386 D6RF19 387 D6RF62 388 D6RF77 389 D6RF86 390 D6RGK8 391D6RGV2 392 D6RJ91 393 D6RJ96 394 D6RJA4 395 D6RJI3 396 D9ZGF4 397 E2DRY6398 E5RGB0 399 E5RGR6 400 E5RGU3 401 E5RH00 402 E5RIV9 403 E6Y3G0 404E7ERL0 405 E7ETE2 406 E7ETF9 407 E7EX41 408 E9PCM2 409 E9PD92 410 E9PFL9411 E9PGT6 412 E9PJF7 413 E9PKD5 414 E9PKP4 415 E9PKW6 416 E9PMR6 417E9PNW4 418 E9PQ82 419 E9PRM1 420 E9PS23 421 E9PS78 422 F5GX11 423 F5GXD8424 F5GZN3 425 F5H520 426 F6MF51 427 F8VUF6 428 FSVYY9 429 F8VZY9 430F8W7P5 431 F8W943 432 FSWBV3 433 F8WCH0 434 F8WD56 435 FSWDR7 436 F8WEQ7437 G3CIH8 438 G3V1B5 439 G3V1S6 440 G3V210 441 G3V281 442 G3V4S5 443G3V5Z7 444 G3XAM7 445 G5E9W1 446 H0Y409 447 H0Y430 448 H0Y465 449 H0Y4A0450 H0Y5U1 451 H0Y7G9 452 H0Y7R7 453 H0Y8M8 454 H0Y9N2 455 H0Y9Q9 456H0YAY4 457 H0YCE1 458 H0YCX6 459 H0YCY8 460 H0YD73 461 H0YEL2 462 H0YGK8463 H0YH82 464 H0YI09 465 H0YI26 466 H0YJP3 467 H0YM36 468 H0YMB1 469H0YMP8 470 H3BPX2 471 H3BQU9 472 H6UK83 473 H6VRG2 474 H7BXY6 475 H7BZL4476 H7C063 477 H7C064 478 H7C0U5 479 H7C1H6 480 H7C1U0 481 H7C2H5 482H7C410 483 H7C5K4 484 H7C5L4 485 H7C5N8 486 H7C5W6 487 H7C5W8 488 I1YAQ5489 I3L1Y9 490 I3L392 491 I3L3P5 492 I3L425 493 I3VM54 494 J3KND3 495J3KNE3 496 J3KPM9 497 J3KRB5 498 J3KRT5 499 J3KRT8 500 J3KSV6 501 J3KTQ0502 J3QKR0 503 J3QL20 504 J3QQT0 505 J3QQX2 506 J3QR48 507 J3QRS3 508J3QS32 509 J3QS41 510 J3QTA5 511 J7HH10 512 J7M3T8 513 K7EJ01 514 K7EJH8515 K7EKM4 516 K7EKW4 517 K7EL50 518 K7ELI6 519 K7EM20 520 K7EP19 521K7EPB9 522 K7ER62 523 K7ERC6 524 K7ESB6 525 K7ESE8 526 K7ESG6 527 K7N7D6528 L7QJ95 529 L8EAR0 530 M0QZ17 531 M0QZC5 532 M0R0N8 533 M0R0Q9 534M0R1H5 535 M1TIV1 536 M1VPF9 537 O00267 538 O00549 539 O14562 540A0A0A0MSA0 541 A0A0R4J2F3 542 B4DHB6 543 B4DHD2 544 B7Z2F4 545A0A0S2Z4D6 546 B4DXI1 547 B4E1U3 548 B7Z2B3 549 B0YJC5 550 B2R4D5 551A0A024R0E5 552 A0A024R151 553 A0A087WZ38 554 B2R7B5 555 B2RDZ9 556B3KN51 557 B4DIJ6 558 B4DNJ0 559 B7Z4P8 560 B7Z5V2 561 B7Z5Z3 562 C9IZA5563 C9J8I8 564 D6RCL1 565 E1P506 566 E5RJR0 567 E7EN95 568 E9PI65 569E9PKA3 570 E9PLE9 571 E9PQ67 572 F5H7C6 573 F5H7T4 574 F5H815 575 G3CC15576 G3V4X5 577 G5E9R9 578 G8I2S9 579 H0Y326 580 H0YAP6 581 H0YDN1 582H0YHV6 583 H0YLD0 584 H0YN28 585 H3BQZ7 586 H3BU13 587 H3BUW1 588 H7BZU1589 H7C1C2 590 H7C1J5 591 H7C248 592 H7C3I4 593 H7C5S0 594 I3L155 595I3L1F7 596 J9ZVQ3 597 K7EL90 598 K7EMN4 599 M0QZ24 600 M0R081 601 O14660602 A0A024R0J4 603 A0A024RDG1 604 A0A024RDW7 605 A0A075B730 606A0A087WTJ7 607 A0A087WU44 608 A0A087WUE6 609 A0A087WZP6 610 A0A0A0MSB2611 A0A0C4DFM8 612 A0A0D9SFN7 613 A0A0S2Z457 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1378 B3KMI7 1379 B4DEW0 1380B4DJN9 1381 B4DQD6 1382 B4DTH9 1383 B4DYS5 1384 B4E1E0 1385 B7Z2B6 1386B7Z4S4 1387 B7Z6T6 1388 B7Z998 1389 B7ZLK3 1390 C9JLV5 1391 C9JTN9 1392D3DPH5 1393 A8K1M2 1394 B0I1S1 1395 B3KN63 1396 B3KQQ9 1397 C9K052 1398E9PLT3 1399 E9PQ34 1400 H0Y3V4 1401 H7C556 1402 H7C5L0 1403 I3L1H5 1404J3KRF5 1405 J3KSI6 1406 J3KSQ2 1407 M0R1M5 1408 A0A0A0MTJ5 1409 B3KWQ61410 E7EXA3 1411 A0A140VKC3 1412 H7BXG7 1413 A8K548 1414 A0A024QZ34 1415A0A087WZ27 1416 A0A0S2Z5H5 1417 A0PJG0 1418 A1L0V1 1419 B2R5H0 1420B3KQM9 1421 B3KV07 1422 B4DEK5 1423 B4DHT9 1424 B4DN99 1425 B4DQ93 1426B4DRN5 1427 B4DUP0 1428 B4DXF3 1429 B4E027 1430 B7Z471 1431 D6RD66 1432H0Y8Q2 1433 H0YKZ6 1434 H7BY97 1435 H7C381 1436 H7C5R6 1437 M0R2L7 1438A0A024QZM6 1439 A0A024R5U5 1440 A0A087WSW9 1441 B3KQQ3 1442 C9JHZ6 1443H0YAS8 1444 H3BR68 1445 Q0Z8D6 1446 Q10567 1447 Q53HA4 1448 Q59EE7 1449Q59F55 1450 Q59GY9 1451 Q59HB9 1452 Q5JRS1 1453 Q5QPQ1 1454 Q5T987 1455Q5U0A0 1456 Q68CX0 1457 Q6DK41 1458 Q6IN67 1459 Q6IT96 1460 Q6MZZ3 1461Q6NS35 1462 Q6P602 1463 Q6P6D7 1464 Q6PJ44 1465 Q12986 1466 Q562S2 1467Q59GK6 1468 Q6PL18 1469 Q6T4R5 1470 A0A024RAV2 1471 A0A087WXC2 1472A4D1M6 1473 A5GZ69 1474 A8K088 1475 A8K0V5 1476 B3KSS4 1477 B3KY38 1478B4DDM1 1479 A0A140VK57 1480 A8K3T7 1481 B2R6X5 1482 B4DX81 1483 C9JIS11484 A0A0R4J2E1 1485 A0A140VJE4 1486 B4DNV6 1487 A0A0A0MRL0 1488A0A0B4J269 1489 A8KAL1 1490 A8MUN2 1495 B2RDY9 1492 B3GQS7 1493 B3KXR41494 B4DLN6 1495 B7Z1Q4 1496 B7Z4R8 1497 B7Z7L7 1498 B9EJB8 1499 C9JIX51500 C9JVS8 1501 D3DUW5 1502 A0A087WTQ3 1503 B4DLR2 1504 B4DT32 1505B4DTV1 1506 B4DX93 1507 B7Z1Y5 1508 B7Z556 1509 C9JD75 1510 A0A024R7A81511 A0A087WT27 1512 A0A140VK58 1513 A5YM51 1514 A8K270 1515 B2RE43 1516B3KRA9 1517 B4DDL4 1518 B4DFW6 1519 B4DHN1 1520 B4DL49 1521 A0A0G2JMS61522 A0A140VJS7 1523 A6YID5 1524 A8K333 1525 B2R918 1526 B4DIE5 1527B4DS71 1528 B4DWW8 1529 B4E357 1530 C9JCK5 1531 D6RAK7 1532 D6RGG3 1533A0A0G2JND4 1534 A8K7Q2 1535 A0A024RDA6 1536 A0A075BTL2 1537 A0A087X1W21538 A0A0D9SFH4 1539 A0A140VK80 1540 A4D1Y2 1541 A6XND9 1542 B3KMV2 1543B3KUH4 1544 B3KWK5 1545 B4E320 1546 C9JB25 1547 D3DSM7 1548 D3VVH7 1549E9PH62 1550 E9PIG4 1551 E9PJR2 1552 F5GZS6 1553 F5H157 1554 F5H5N6 1555F6XY72 1556 F8VX58 1557 F8W1K6 1558 F8W6C1 1559 F8WD80 1560 F8WDD7 1561G3V4Q1 1562 G5EA09 1563 H0UI22 1564 H0Y5J5 1565 H0Y8H4 1566 H0YAI1 1567H0YCQ2 1568 H0YDJ9 1569 H0YDP9 1570 H0YEB7 1571 H0YFT8 1572 H0YNV5 1573H7C457 1574 I3V9V3 1575 I6L984 1576 J3KS15 1577 K7EJJ5 1578 K7EQX3 1579B3KR26 1580 B4DVD7 1581 A0A087WWW9 1582 A0A140T9B7 1583 B1APP6 1584B2R960 1585 B4E2F9 1586 B7Z582 1587 F5GY34 1588 F8VZH8 1589 G3V361 1590H0YB61 1591 H0YBH9 1592 H0YD93 1593 H7BXZ5 1594 H7C3V5 1595 K7EM73 1596A8K8D9 1597 A8K8S6 1598 B3KWT5 1599 B4E0H6 1600 H7BXV5 1601 I3L3X7 1602B7ZLZ7 1603 F8W692 1604 F8WC54 1605 H0UI83 1606 H0YA75 1607 H0YI37 1608A0A024R0A9 1609 A8K912 1610 B4DH11 1611 A0A024R4I7 1612 A0A024R688 1613A0A024R6D1 1614 A0A024RC87 1615 A0A087X0M0 1616 A0A0A0MS98 1617A0A0U1RRM8 1618 A0A140VJM1 1619 A1A5B0 1620 A8K9E1 1621 A8KA75 1622B2R7T8 1623 B2R995 1624 B3KND4 1625 B3KSM5 1626 B3KXY0 1627 B4DJS0 1628B4DM61 1629 B4DMH3 1630 B4DPS8 1631 B4DPX8 1632 B4DPZ4 1633 B4DTH4 1634B4DTN7 1635 B4DXW2 1636 B4DYM0 1637 B4DZB4 1638 B4DZP5 1639 B4E0L1 1640B4E1R7 1641 B7Z1F8 1642 B7Z4V4 1643 B7Z577 1644 B7Z9L0 1645 C9J296 1646C9JTX5 1647 E5RG28 1648 E9PJ32 1649 E9PQ98 1650 F5H136 1651 F8VZB4 1652F8WD59 1653 F8WEB3 1654 G3V3U4 1655 G9K388 1656 H0YEP3 1657 H0YFX4 1658H0YKV8 1659 H0YLS6 1660 H7BY18 1661 H7C144 1662 H7C2W6 1663 J3KNL1 1664M0QZG6 1665 M0R3C9 1666 P29279 1667 Q0P5U8 1668 Q14215 1669 Q14DR2 1670Q1RMC8 1671 Q2TU64 1672 Q2VIM7 1673 Q4ZF95 1674 Q504U3 1675 Q53FR4 1676Q53GR3 1677 Q53H39 1678 Q56CY1 1679 Q59G97 1680 Q59H37 1681 Q5JVS8 1682Q5SX86 1683 Q5SXM2 1684 Q5T0S3 1685 Q5U0B9 1686 Q5VVQ1 1687 Q6DC98 1688Q6NSG7 1689 Q6PJT4 1690 A0A024R060 1691 A0A024R498 1692 A0A024R599 1693A0A024R8B2 1694 A0A024R9F3 1695 A0A024R9Y3 1696 A0A087WTP5 1697A0A087WU93 1698 A0A087WW43 1699 A0A087WXX1 1700 A0A087WYV8 1701A0A087WYX9 1702 A0A087X080 1703 A0A087X271 1704 A0A087X2I4 1705A0A090N7V0 1706 A0A0A0MR56 1707 A0A0B4J1R7 1708 A0A0B6XK25 1709A0A0C4DFW8 1710 A0A0D9SET8 1711 A0A0D9SG79 1712 A0A0G2JPA8 1713A0A0J9YWE8 1714 A0A0J9YX86 1715 A0A0K0Q0G7 1716 A0A0R4J2G7 1717A0A0S2Z3X3 1718 A0A0S2Z3X8 1719 A0A0U1RR07 1720 A0A126GWA0 1721A0A140T936 1722 A0A140VJT0 1723 A0A140VK45 1724 A0A173GMX4 1725 A4D1J91726 A4QPA1 1727 A4UCT1 1728 A4UCT9 1729 A5YM53 1730 A6NDY9 1731 A8K1S01732 A8K2I0 1733 A8K477 1734 A8K4G9 1735 A8K590 1736 A8K5T0 1737 A8K6901738 A8K6S3 1739 A8K7F7 1740 ASK7Q1 1741 A8K7T8 1742 A8K8A4 1743 A8K9001744 A8K979 1745 A8MXT2 1746 B0YJC4 1747 B2R932 1748 B2RBP3 1749 B3KMH61750 B3KMM7 1751 B3KNB9 1752 B3KNF2 1753 B3KNF9 1754 B3KRK8 1755 B3KT981756 B3KTM1 1757 B3KU27 1758 B3KU60 1759 B3KUY5 1760 B3KX16 1761 B3W6H41762 B4DG55 1763 B4DG73 1764 B4DGT1 1765 B4DGX9 1766 B4DI19 1767 B4DID61768 B4DIW2 1769 B4DJ06 1770 B4DJ44 1771 B4DMJ0 1772 B4DN77 1773 B4DND61774 B4DQQ2 1775 B4DQX0 1776 B4DQZ0 1777 B4DSF8 5778 B4DT18 1779 B4DW311780 B4DX43 1781 B4DYA7 1782 B4DZX4 1783 B4E392 1784 B4E3Q9 1785 B5MDS31786 B7Z5H9 1787 B7Z6E5 1788 B7Z6L5 1789 B7Z7W6 1790 B7Z822 1791 B7Z8421792 B7Z8E7 1793 B7ZAE8 1794 B7ZAP6 1795 B7ZAX6 1796 B7ZKR5 1797 C6GLZ11798 C9J7B7 1799 C9JBU9 1800 C9JIF9 1801 C9JMZ3 1802 C9JQJ4 1803 C9JSL41804 C9JT74 1805 C9JU11 1806 C9K0H9 1807 D0V481 1808 D3DT96 1809 D3DUT51810 D6R904 1811 D6RJI2 1812 E0Z3H2 1813 E2QRI1 1814 E5RG57 1815 E5RGA21816 E7EPM4 1817 E7EU13 1818 E7EUI6 1819 E9PCC8 1820 E9PF58 1821 E9PL371822 E9PLD0 1823 E9PLK3 1824 E9PLY5 1825 E9PMN5 1826 E9PPQ4 1827 E9PQK61828 E9PRR2 1829 F1BXA6 1830 F1D8R6 1831 F5H6W8 1832 F8VP67 1833 F8VWV41834 F8WE98 1835 G3V2E8 1836 G3V2W4 1837 G3V380 1838 G3V393 1839 G3V5111840 G3XL79 1841 G5E9Z2 1842 H0Y400 1843 H0Y897 1844 H0YAB0 1845 H0YAH81846 H0YBW3 1847 H0YCK7 1848 H0YEG8 1849 H0YEP8 1850 H0YET5 1851 H0YGZ31852 H0YI33 1853 H0YKU5 1854 H0YLD1 1855 H0YLJ1 1856 H0YLM2 1857 B3BN341858 H3BN75 1859 H3BNR4 1860 H3BNT7 1861 H3BPG7 1862 H3BQA8 1863 H3BRL21864 H3BRV9 1865 H3BU24 1866 H3BUU9 1867 H3BUV8 1868 H3BV30 1869 H7BZE91870 H7C0M5 1871 H7C0V9 1872 H7C278 1873 H7C531 1874 H7C543 1875 H7C5J51876 I3L2R7 1877 I3L3Q7 1878 I3L3Z3 1879 I3V9T1 1880 J3QS16 1881 J3QTJ61882 K4RH61 1883 K7ENH5 1884 K7EPG3 1885 K7ERA3 1886 K7ERX2 1887 L8E7581888 A0A087WT80 1889 A0A087WUR8 1890 A0A024RA94 1891 A0A087WYP2 1892A0A096LPA6 1893 A0A0A1HAN9 1894 A0A0C4DGH2 1895 A0A0G2JP90 1896A0A0S2Z3H5 1897 A0A0S2Z476 1898 A0A0U1RRI8 1899 A0A0U5BLD0 1900A0A0U5EM55 5901 A0A140VK69 1902 A0A140VK70 1903 A8K309 1904 A8K3Q7 1905A8K4W8 1906 A8K8U1 1907 A8K9X5 1908 A8MV29 1909 A8YQF3 1910 B2RD14 1911B2RDI5 1912 B3KQM1 1913 B3KRK1 1914 B3KXF2 1915 B4DFF9 1916 B4DFG7 1917B4DHC3 1918 B4DHQ7 1919 B4DJQ5 1920 B4DMB6 1921 B4DMT4 1922 B4DN21 1923B4DN30 1924 B4DN32 1925 B4DN78 1926 B4DNM8 1927 B4DPP0 1928 B4DQK5 1929B4DR44 1930 B4DRF7 1931 B4DRX1 1932 B4DSD3 1933 B4DSQ5 1934 B4DUK1 1935B4DUM2 1936 B4DVL8 1937 B4DWA8 1938 B4DXJ6 1939 B4DYU2 1940 B4E163 1941B4E190 1942 B4E1H9 1943 B4E2V8 1944 B4E356 1945 B5A941 1946 B7Z237 1947B7Z6I3 1948 B7Z766 1949 B7Z9C4 1950 B7ZAB0 1951 C6ZGX2 1952 C9JAM8 1953C9JEG3 1954 C9K0C5 1955 D3DPU0 1956 D6REX0 1957 D6RFX4 1958 D6RIR7 1959E5KRG5 1960 E5RFS1 1961 E9PK25 1962 E9PKK9 1963 E9PMR4 1964 E9PNW5 1965E9PQH6 1966 F5GYU2 1967 F8VP03 1968 G1UD79 1969 H0YA83 1970 H0YBG6 1971H0YIK3 1972 H0YMD9 1973 H0YN52 1974 H3BLS7 1975 H7C183 1976 I3L0S0 1977I3L3P9 1978 I3L3W3 1979 I4AY87 1980 J3KQ66 1981 J3KTI8 1982 J3QKQ5 1983J3QT34 1984 J9R021 1985 K7EP68 1986 L7RSL3 1987 M0R0W6 1988 M0R2Z9 1989O14549 1990 O15018 1991 O15289 1992 P07195 1993 P0C221 1994 P11217 1995P17032 1996 P18669 1997 P21912 1998 P47871 1999 P48741 2000 P53675 2001P61201 2002 Q0PNF2 2003 Q14329 2004 Q14769 2005 Q1HA41 2006 Q1LZN2 2007Q2I0A6 2008 Q2PUK1 2009 Q2V4X9 2010 Q2VPA1 2011 Q3B787 2012 Q3MIC3 2013Q4G1B8 2014 Q4JCP9 2015 Q4LE33 2016 Q4LE71 2017 Q4V347 2018 Q4W5E0 2019Q53F79 2020 Q53FX5 2021 Q53G35 2022 Q53G83 2023 Q53GG3 2024 Q53GZ2 2025Q53HB6 2026 Q53RS1 2027 Q53SM6 2028 Q53TA7 2029 Q53TY1 2030 053X91 2031Q562U1 2032 Q562X2 2033 Q56VW8 2034 Q58I23 2035 Q59E90 2036 Q59EB3 2037Q59EL5 2038 Q59F04 2039 Q59FC6 2040 Q59FD0 2041 Q59FF0 2042 Q59FJ0 2043Q59FK6 2044 Q59GI7 2045 Q59GI8 2046 Q59GL1 2047 Q59GS3 2048 Q59H74 2049Q59HA6 2050 Q5CZ99 2051 Q5JPB8 2052 Q5JPE4 2053 Q5JTH9 2054 Q5QNZ2 2055Q5SUI8 2056 Q5T3N0 2057 Q5TA01 2058 Q5TBG5 2059 Q5TEK2 2060 Q5TZP0 2061Q68D23 2062 Q69YT6 2063 Q6DHY5 2064 Q6FGL0 2065 Q6FHZ0 2066 Q6FI97 2067Q6I7N8 2068 Q6IB71 2069 Q6IC76 2070 Q6IPN0 2071 Q6LCH2 2072 Q6MZF4 2073A0A024R411 2074 A0A024R3H2 2075 A0A024R3V8 2076 A0A024R5L0 2077A0A024RAM4 2078 A0A087WYF2 2079 A0A097EV82 2080 A0A0G2JRF9 2081A0A125S6H6 2082 A0A140VK56 2083 A0PJ81 2084 A8K8B0 2085 A8KAK1 2086A8MXH5 2087 B2R8G3 2088 B2R921 2089 B4DE40 2090 B4DIX1 2091 B4DSW9 2092B4DX08 2093 B4E0B4 2094 B4E0X6 2095 B5MD23 2096 B7Z2Z0 2097 B7Z3Z2 2098B7Z4G0 2099 B7ZMJ0 2100 C9JHG2 2101 C9JK86 2102 C9JKQ7 2103 C9JPC3 2104D6RIA0 2105 E5RJ43 2106 E9PRU1 2107 F5GXU1 2108 F8WAA0 2109 H0Y5C6 2110H0Y875 2111 H0Y9P0 2112 H0YAF9 2113 H0YIB2 2114 H0YN01 2115 H7C3F9 2116H7C4Y7 2117 J3QRA5 2118 J3QS88 2119 P19022 2120 Q05BR9 2121 Q14568 2122Q53SM9 2123 Q59F65 2124 Q5FWG8 2125 Q6FH10 2126 Q6M1B8 2127 Q6MZT4 2128Q6NZ44 2129 A0A024R084 2130 A0A024R5W3 2131 A0A024R6B5 2132 A0A024RDG62133 A0A024RDY3 2134 A0A087X0M6 2135 A0A0A0MRQ5 2136 A0A0U1RR39 2137A0A140VJR3 2138 A7MBM6 2139 A8K1Y7 2140 A8K781 2141 B3KUE5 2142 B3KVI82143 B4DQH3 2144 B4DRT3 2145 B4DWQ5 2146 B4DY05 2147 B4DY98 2148 F5H2L32149 F8W787 2150 H0YMC2 2151 H7C2S8 2152 Q14918 2153 Q49A43 2154 Q4W5D32155 Q53FG3 2156 Q53H18 2157 Q53HJ0 2158 Q59ET0 2159 F6S8N6 2160 F6X2W22161 H0Y8I3 2162 H0YCR7 2163 H0YMM1 2164 J3KQ42 2165 M1LAK4 2166 Q435322167 Q0P682 2168 Q59FU7 2169 Q59GT2 2170 Q5T4U8 2171 Q5T9S5 2172 Q5TCI82173 A0A024R7C7 2174 A0A087WWU3 2175 A0A0A0MSV3 2176 A0A140GPP7 2177A0A140VJX3 2178 A2VCK8 2179 A7E2S2 2180 B2R708 2181 B3KPG0 2182 B3KQK12183 B3KTJ9 2184 B3KWY2 2185 B4DDG4 2186 B4DMK2 2187 B4DP06 2188 B4DT732189 B4DU71 2190 B7Z4C7 2191 B9VPB4 2192 C9JPC0 2193 C9JUU5 2194 C9K0552195 D3DSM4 2196 E7ER45 2197 E9PCH4 2198 E9PN91 2199 E9PNX1 2200 F6XZQ72201 F8WC37 2202 G3V295 2203 H0UI49 2204 H0Y4E8 2205 H0Y789 2206 H0Y8F12207 H0YMJ5 2208 H3BV48 2209 H7C3T2 2210 A0A024QYX7 2211 A0A024R017 2212A0A024R1D0 2213 A0A024R2W4 2214 A0A024R321 2215 A0A024R4K5 2216A0A024R7J0 2217 A0A024R8K6 2218 A0A024R8Q1 2219 A0A024R8U1 2220A0A024RAB2 2221 A0A024RAJ6 2222 A0A024RCA7 2221 A0A024RDW8 2224A0A075EKM8 2225 A0A087WT21 2226 A0A087WU05 2227 A0A087WW79 2228A0A087WWP7 2229 A0A087WWY3 2230 A0A0S7WY61 2231 A0A087WZM5 2232A0A087WZN9 2233 A0A087X1J2 2234 A0A0A0MQX7 2235 A0A0A0MRF9 2236A0A0A0MRH2 2237 A0A0A0MTC1 2238 A0A0A7M1X5 2239 A0A0B6XK12 2240A0A0C4DFS1 2241 A0A0G2JJ52 2242 A0A0G2JN90 2243 A0A0G2JNE7 2244A0A0G2JQ91 2245 A0A0S2Z487 2246 AGA0S2Z4T2 2247 A2RUE7 2248 A6XMV8 2249A6YID7 2250 A8K0T9 2251 A8K126 2252 A8K168 2253 A8K335 2254 A8K6D3 2255A8K849 2256 B0I1S3 2257 B3KS68 2258 B3KS71 2259 B3KT0 2260 B3KU58 2261B4DDF3 2262 B4DE78 2263 B4DF49 2264 B4DG47 2265 B4DHV7 2266 B4DJJ9 2267B4DMB5 2268 B4DNH1 2269 B4DP69 2270 B4DPW9 2271 B4DTA8 2272 B4DTH2 2273B4DYM7 2274 B4E1B0 2275 B4E284 2276 B7Z2F0 2277 B7Z2I6 2278 B7Z597 2279B7Z754 2280 B7Z924 2281 B7Z9B8 2282 B7ZA28 2283 B7ZAG1 2284 B7ZAY4 2285B7ZML4 2286 C9IY79 2287 C9JKZ2 2288 D6RHZ5 2289 D9ZGF8 2290 E2ITE0 2291E5RGZ4 2292 E5RH50 2293 E5RII0 2294 E5RJ14 2295 E5RK62 2296 E5RK63 2297E7EMB3 2298 E7EMF1 2299 E9KL30 2300 E9PD53 2301 E9PIA9 2302 E9PJF9 2303E9PKH0 2304 E9PM19 2305 E9PM22 2306 E9PMC9 2307 E9PNQ9 2308 E9PPD2 2309E9PPK9 2310 E9PPR9 2311 E9PQ70 2312 E9PR84 2313 E9PRJ8 2314 F2Z3H1 2315F4ZW63 2316 F5GWI0 2317 F5H3N3 2318 F5H4N4 2319 F5H4Z8 2320 F8VR50 2321F8VRX1 2322 F8VXW2 2323 F8W1A0 2324 F8W1H8 2325 F8W1K5 2326 F8W9J4 2327F8WAE6 2328 F8WD26 2329 F8WDY7 2330 G1EPL1 2331 G3V1E2 2332 G3V2H3 2333G3V2X9 2334 G3V4U0 2335 G3V5X8 2336 G5EA44 2337 G8XWS8 2338 H0UI76 2339H0Y351 2340 H0Y3A8 2341 H0Y4K8 2342 H0Y4R1 2343 H0Y554 2344 H0Y586 2345H0Y5C0 2346 H0Y5F3 2347 H0Y5U0 2348 H0Y8S0 2349 H0Y987 2350 H0Y998 2351H0Y9C2 2352 H0YAM7 2353 H0YAP9 2354 H0YC35 2355 H0YC94 2356 H0YCR9 2357H0YEM3 2358 H0YET2 2359 H0YIH3 2360 H0YJ03 2361 H0YJD3 2362 H0YKB3 2363H0YKS8 2364 H0YL50 2365 H0YM03 2366 H0YM92 2367 H3BPF6 2368 H3BTD4 2369H3BTN5 2370 H3BU78 2371 H3BUH7 2372 H7BY82 2373 H7C1M6 2374 H7C2H7 2375H7C2Q8 2376 H7C2U0 2377 H7C2Y0 2378 H7C4X4 2379 I3L4F9 2380 I3L4N7 2381J3K000 2382 K7EKL3 2383 K7EKS1 2384 K7EM90 2385 K7EMN2 2386 K7EPL0 2387K7ERF7 2388 M0R0U4 2389 M1VKI9 2390 O94985 2391 O95617 2392 P30419 2393P98174 2394 Q00839 2395 Q05BW8 2396 Q05DA4 2397 Q05DS5 2398 Q14650 2399Q1RMZ5 2400 Q2EF79 2401 Q2VP91 2402 Q2YD88 2403 Q3B7A7 2404 Q4G0Q4 2405Q4LE57 2406 Q4VC00 2407 Q504V9 2408 Q508I7 2409 Q53F09 2410 Q53FI4 2411Q53FK2 2412 Q53FU5 2413 Q53GU8 2414 Q53GW3 2415 Q53HE2 2416 Q53HU7 2417Q53S41 2418 Q58FF2 2419 Q59EG5 2420 Q59EZ1 2421 Q59F71 2422 Q59FD9 2423Q59GH5 2424 Q59GK2 2425 Q59GZ8 2426 Q59HA3 2427 Q59HB3 2428 Q59HB5 2429Q5FC05 2430 Q5HYA2 2431 Q5JPH6 2432 Q5JQ13 2433 Q5QNY5 2434 Q5T0F3 2435Q5TA02 2436 Q5TFM2 2437 Q60FE5 2438 Q658J0 2439 Q68CW0 2440 Q68CX6 2441Q6AI13 2442 Q6EHZ3 2443 Q6PEG1 2444 Q6PK56 2445 Q6PKD2 2446 Q6PQ81 2447Q6S4P3 2448 Q6U2L6 2449 Q6U8A4 2450 Q6UQL6 2451 Q6UUU9 2452 Q6W6M8 2453Q6XYD2 2454 Q6ZMY0 2455 Q6ZMY3 2456 Q6ZN49 2457 Q6ZNB8 2458 Q6ZNL4 2459Q6ZNU3 2460 Q6ZP35 2461 Q6ZP53 2462 Q6ZR44 2463 Q6ZRZ8 2464 Q6ZS01 2465Q6ZS03 2466 Q6ZS14 2467 Q6ZSD7 2468 Q6ZSL4 2469 Q6ZT31 2470 Q6ZT94 2471Q6ZTY7 2472 Q6ZVC6 2473 Q71RC2 2474 Q71S06 2475 Q71SW6 2476 Q71UD4 2477Q71UM7 2478 Q75MN1 2479 Q75MY0 2480 Q75N18 2481 Q75N89 2482 Q7KZ71 2483Q7L4M3 2484 Q7L4N0 2485 Q7L8K0 2486 Q7RTQ9 2487 Q7RU04 2488 Q7Z2F6 2489Q7Z2X7 2490 Q7Z355 2491 Q7Z3G7 2492 Q7Z3T9 2493 Q7Z427 2494 Q7Z487 2495Q7Z497 2496 Q7Z4Z1 2497 Q7Z6M3 2498 Q7Z738 2499 Q7Z757 2500 Q7Z7K9 2501Q7Z7R0 2502 Q86TQ3 2503 Q86TX4 2504 Q86U12 2505 Q86U75 2506 Q86U79 2507Q86UP3 2508 Q86V48 2509 Q86W61 2510 Q86WD0 2511 Q86XU5 2512 Q86YN0 2513Q8IVF4 2514 Q8IWC8 2515 Q8IWL5 2516 Q8IXX0 2517 Q8IY44 2518 Q8IY97 2519Q8IY98 2520 Q8IYQ9 2521 Q8IZD4 2522 Q8N1I1 2523 Q8N1I8 2524 Q8N274 2525Q8N294 2526 QSN4Z1 2527 Q8N505 2528 Q8N5L9 2529 Q8N6NS 2530 Q8N6P3 2531Q8N7Y3 2532 Q8N7Z5 2533 Q8N9C4 2534 Q8N9F8 2535 Q8N9K4 2536 Q8N9Q8 2537Q8NA68 2538 Q8NB89 2539 Q8NBH6 2540 Q8NCJ3 2541 Q8NE02 2542 Q8NF03 2543Q8NF19 2544 Q8NF24 2545 Q8NF60 2546 Q8NG20 2547 Q8NH73 2548 Q8NI27 2549Q8TAS1 2550 Q8TAT5 2551 Q8TB01 2552 Q8TB95 2553 Q8TBN2 2554 Q8TDG6 2555Q8TDS4 2556 Q8TEP3 2557 Q8TES4 2558 Q8WTY5 2559 Q8WU03 2560 Q8WUI6 2561Q8WVX2 2562 Q8WW96 2563 Q8WWB2 2564 Q8WX69 2565 Q8WZ56 2566 Q92468 2567Q92547 2568 Q92681 2569 Q92945 2570 Q93063 2571 Q93093 2572 Q969I5 2573Q96AA2 2574 Q96AQ0 2575 Q96AR9 2576 Q96AX1 2577 Q96B07 2578 Q96B60 2579Q96BA4 2580 Q96BG6 2581 Q96C61 2582 Q96CD8 2583 Q96CV8 2584 Q96D30 2585Q96DQ5 2586 Q96DZ4 2587 Q96EB3 2588 Q96G38 2589 Q96GF5 2590 Q96GW1 2591Q96HC2 2592 Q96HF4 2593 Q96HI1 2594 Q96HN5 2595 Q96IE3 2596 096JJ7 2597Q96K48 2598 Q96K89 2599 Q96KC0 2600 Q96LJ7 2601 Q96LN7 2602 Q96LR2 2603Q96MN8 2604 Q96N76 2605 Q96NB3 2606 Q96NX2 2607 Q96PA3 2608 Q96PJ0 2609Q96PV0 2610 Q96Q06 2611 Q96QU5 2612 Q96RL8 2613 Q96RP4 2614 Q96RS2 2615Q96RZ7 2616 Q96SE4 2617 Q99435 2618 Q99529 2619 Q99538 2620 Q99666 2621Q9BR56 2622 Q9BR60 2623 Q9BS14 2624 Q9BS75 2625 Q9BSD0 2626 Q9BTL0 2627Q9BVS9 2628 Q9BW34 2629 Q9BXG2 2630 Q9BXV5 2631 Q9BZ93 2632 Q9BZQ0 2633Q9H049 2634 Q9H0M4 2635 Q9B2E0 2636 Q9H2E1 2637 Q9H369 2638 Q9H3B0 2639Q9H3R3 2640 Q9H3U3 2641 Q9H511 2642 Q9H5S1 2643 Q9H5T0 2644 Q9H717 2645Q9H7J0 2646 Q9H8J8 2647 Q9HAP0 2648 Q9HAP1 2649 Q9HB74 2650 Q9HBB2 2651Q9HBP0 2652 Q9HC77 2653 Q9HCI6 2654 Q9NP01 2655 Q9NPA9 2656 Q9NPM2 2657Q9NQG5 2658 Q9NRE2 2659 Q9NRY4 2660 Q9NS89 2661 Q9NSH2 2662 Q9NSK3 2663Q9NSM5 2664 Q9NT11 2665 Q9NTC4 2666 Q9NTU6 2667 Q9NV59 2668 Q9NVF8 2669Q9NVY6 2670 Q9NW05 2671 Q9NW43 2672 Q9NWD6 2673 Q9NWP5 2674 Q9NWW3 2675Q9P0V0 2676 Q9P1C5 2677 Q9P1G4 2678 Q9P1N9 2679 Q9P1Y0 2680 Q9P262 2681Q9P2G7 2682 Q9UBC7 2683 Q9UD42 2684 Q9UD69 2685 Q9UDE8 2686 Q9UDY3 2687Q9UDZ8 2688 Q9UE33 2689 Q9UE89 2690 Q9UES0 2691 Q9UFZ4 2692 Q9UG85 2693Q9UGU0 2694 Q9UIU0 2695 Q9UJ56 2696 Q9UJM0 2697 Q9UJM1 2698 Q9UJN9 2699Q9UJZ2 2700 Q9UJZ7 2701 Q9UM89 2702 Q9UMB3 2703 Q9UMN4 2704 Q9UNF3 2705Q9UNU2 2706 Q9UPH5 2707 Q9UQC1 2708 Q9UQS6 2709 Q9Y267 2710 Q9Y2Q2 2711Q9Y4R1 2712 Q9Y546 2713 Q9Y5E1 2714 Q9Y623 2715 Q9Y6D3 2716 Q9Y6N6 2717Q9Y6U3 2718 R4GMY1 2719 R4GNC2 2720 R4GNC7 2721 R4RWV3 2722 S4R328 2723S4R3E9 2724 S4R3G7 2725 S4R451 2726 S5FMV1 2727 U3KPX5 2728 U3KQA9 2729U3KQE2 2730 U3KQI3 2731 U3KQK2 2732 U3KQK8 2733 U3KQP1 2734 U3PXP0 2735U3REJ1 2736 V5QSK8 2737 V9GYK3 2738 V9GYL0 2739 V9GYV7 2740 V9GZ17 2741V9GZ54 2742 V9GZ76 2743 V9GZR9 2744 V9H0H3 2745 V9H1C1 2746 V9H1D9 2747V9HVZ6 2748 V9HVZ7 2749 V9HW24 2750 V9HW31 2751 V9HW35 2752 V9HW55 2753V9HW63 2754 V9HW80 2755 V9HW89 2756 V9HW90 2757 V9HW95 2758 V9HWA6 2759V9HWA9 2760 V9HWC0 2761 V9HWC1 2762 V9HWC6 2763 V9HWC7 2764 V9HWE9 2765V9HWF4 2766 V9HWF5 2767 V9HWG9 2768 V9HWH6 2769 V9HWH9 2770 W5X314 2771W6I206 2772 W6MEN4 2773 W8QEH3 2774 W8QRJ0 2775 X5D2F4 2776 X5D2Z4 2777X5D784 2778 X5D7K9 2779 X5D7R7 2780 X5DQS5 2781 X5DQV1 2782 X5DR74 2783X6R3R3 2784 X6R6Z1 2785 X6RJP6 2786 X6RLX0

TABLE 5 Proteins in both NPEX and MSCEX 1 A0A024RBF5 2 A0A087WTU5 3A0A087WTY5 4 A0A087WZY3 5 A0A0A0MSD7 6 A0A0U1RQQ9 7 A8K457 8 B0YIW5 9B2R6M6 10 B2RWN5 11 B3KM34 12 B3KS32 13 B4DK21 14 B4DLA8 15 B4DM04 16B4DYP3 17 B7Z220 18 B7Z3F8 19 A0A0A0MRE5 20 A8K5A6 21 B2RWN8 22 B3KQE623 B4DIY9 24 F2Z2Y4 25 I7GY12 26 K7EIZ7 27 A1DRY3 28 A8K2S5 29 B4DJ85 30D3DSZ2 31 D6RHJ6 32 E5RK22 33 A0A090N7W7 34 A0A024R9U8 35 A0A087WTQ1 36A0A0A0MTS5 37 A0A0J9YWF7 38 A0A0S2Z5A6 39 A0A140G945 40 A0PJA6 41 A8MYA242 B3KY01 43 B4DDZ2 44 B4DJ87 45 B4DN40 46 B4DPI7 47 B4DQJ6 48 B7WNR7 49B7ZAV4 50 C9J4J0 51 D1CS68 52 E9PR32 53 H0Y4V9 54 H0YE86 55 H3BM13 56H7C124 57 J3KNF5 58 J3KTE8 59 J3QQZ1 60 K7EMU5 61 A0A087X1K6 62 B9EK6563 D2CPJ9 64 A8K9G8 65 E9PDY4 66 E9PKB7 67 F5GWB9 68 H0Y5A3 69 H0YDX7 70H0YGC5 71 I3L2H2 72 K7ENM8 73 A0A024R2Q7 74 A0A024RAC9 75 A0A087X043 76A1L497 77 A8K5C2 78 B2R7A1 79 B3KML1 80 B4DPN0 81 B4DPP6 82 B4DSG6 83B4DW61 84 B4DY21 85 B4E1C1 86 B7Z3E5 87 B7Z8R2 88 B7ZM83 89 C9JUF9 90A0A0S2SW46 91 A0A140G961 92 A5YM44 93 A8K3I0 94 B2RCM2 95 B2RD90 96B2RDH6 97 B2RUU6 98 B3KPC9 99 B4DKV6 100 C7FEB0 101 E7EUN2 102 F2Z2C0103 G3F4G3 104 H3BUZ9 105 I3L2G4 106 J3KST4 107 L0R875 108 M0QY24 109M0QY93 110 M0R167 111 A0A140T9N1 112 B3KV66 113 B2RN10 114 B4DX19 115A0A087WTM7 116 A0A087WVZ9 117 A0A087WZI7 118 A0A0C4DGZ0 119 A0A0F7T737120 A0A126GW30 121 A8K5R3 122 B1AA16 123 B2RD71 124 B4DQ03 125 B4DRU3126 B4E1H3 127 B4E3L4 128 B5A930 129 G1UI21 130 H7C5E8 131 I3L0S6 132K7ELE6 133 K7ERU1 134 O00303 135 O15232 136 O75144 137 O75643 138 O95757139 O95816 140 P00390 141 P04406 142 P05455 143 P06576 144 P11908 145P12277 146 P13010 147 P13797 148 P13798 149 P14625 150 P17787 151 P18206152 P20592 153 P20618 154 P21399 155 P23381 156 P24534 157 P25787 158P30520 159 P30566 160 P35052 161 P37837 162 P40925 163 P43490 164 P43699165 P48723 166 P49721 167 P49916 168 P50991 169 P55290 170 P60842 171P61158 172 P61970 173 P78357 174 P78371 175 Q02809 176 Q04446 177 Q05BW9178 Q05CU5 179 Q05DQ6 180 Q08431 181 Q13191 182 Q14397 183 Q14974 184Q16555 185 Q4G0U7 186 Q4LE70 187 Q4QQP8 188 Q56A80 189 Q58F05 190 Q59GL5191 Q59H14 192 Q5SRE5 193 Q5VTG7 194 Q6P1A2 195 Q6P668 196 B4DVZ8 197E9PN76 198 A0A0A0MS56 199 A8K228 200 B4DHL3 201 A0A024R0V9 202A0A024R2F7 203 B2RB99 204 B3KVJ8 205 B4DL71 206 B7Z5Q5 207 F8WCB0 208G3V470 209 H0YGP2 210 H7BXX0 211 P11310 212 P13639 213 P22234 214 P41250215 P52209 216 P98095 217 Q00610 218 Q04760 219 Q1JQ81 220 Q4LE34 221Q4ZG60 222 Q562P0 223 Q5SZP4 224 Q5T7Q5 225 Q6NR85 226 Q6PKB2 227A0A024RDI0 228 B3KVT0 229 B3KW08 230 B5BU64 231 F8VTS6 232 Q17RV3 233Q4LE35 234 Q53ZD9 235 H7BZ78 236 P20700 237 P23528 238 P28066 239 P43686240 P51665 241 P63241 242 Q12765 243 Q14666 244 Q5T9B7 245 A0A024R0F1246 A6NNK5 247 A7Y9J9 248 B2RCN5 249 B3KNG8 250 B3KW67 251 B4DJ43 252B7ZBJ6 253 E5RGB1 254 E9PNV8 255 G5E9C5 256 H0YD68 257 O15020 258A0A024R3W8 259 A0A024R4E7 260 A0A024R7L2 261 A0A024R8K8 262 A0A024R8L5263 A0A024R8U4 264 A0A024R9F1 265 A0A087WVC6 266 A0A087WW67 267A0A087WYA1 268 A0A0C4DFY7 269 A0A0G2JNI0 270 A0A0J9YY10 271 A0A126GVS6272 A2RTX5 273 A6NF36 274 A8K324 275 A8K4N3 276 A8K8J5 277 A8K8R2 278B3KQ44 279 B3KWP3 280 B3KXW2 281 B4DEL6 282 B4DH82 283 B4DJT2 284 B4DLC6285 B4DNG2 286 B4DS99 287 B4DWK2 288 B4DYK9 289 B4DYX8 290 B4E303 291B4E367 292 B7Z1P9 293 B7Z671 294 C9JDR5 295 C9JVG0 296 D3DTQ7 297 D3DXG8298 D4PHA4 299 E3W983 300 E5G743 301 E5RG96 302 E5RGT3 303 E7EVQ3 304E9JVC4 305 E9PH60 306 E9PK12 307 F5GXY0 308 F5GY37 309 F5H2D1 310 F5H8J8311 F8UU18 312 F8VWL0 313 F8W7F7 314 G3V5M4 315 H0Y3H6 316 H0Y6A5 317H0YBF7 318 H0YF13 319 H0YGG5 320 H0YL42 321 H3BRY5 322 H7C463 323 I6NLS4324 J3QKX5 325 O00410 326 O14943 327 O15067 328 O95996 329 O96005 330P05386 331 P06744 332 P08069 333 P12956 334 P22102 335 P29401 336 P30153337 P32119 338 P42785 339 P50747 340 P53396 341 P62314 342 Q06323 343Q06AH7 344 Q08629 345 Q13393 346 Q13616 347 Q14563 348 Q15828 349 Q1KLZ0350 Q29RF7 351 Q210A9 352 Q32Q12 353 Q495G0 354 Q53R19 355 Q59E15 356Q59ET3 357 Q59GA8 358 Q5SYQ9 359 Q5XUU0 360 Q6FH47 361 Q6IFB0 362 Q6LAP8363 Q6W4X9 364 Q6XYC2 365 Q6ZMN7 366 Q6ZS81 367 Q6ZTQ4 368 Q6ZWK7 369Q7L2H7 370 Q7Z3Y4 371 Q7Z3Z0 372 Q7Z442 373 Q7Z4C2 374 Q7Z534 375 Q7Z5C1376 Q7Z6C1 377 Q86T01 378 Q86T64 379 Q86VU1 380 Q86X45 381 Q86XV5 382Q86YS3 383 Q8IVC0 384 Q8IVV2 385 Q8IWJ2 386 Q8IXV0 387 Q8IYT4 388 Q81Z41389 Q8IZC6 390 Q8N303 391 Q8N3N3 392 Q8N3T6 393 Q8N696 394 Q8N8W4 395Q8NAD5 396 Q8NBX0 397 Q8NFN8 398 Q8NG11 399 Q8TA92 400 Q8TAK2 401 Q8TCE1402 Q8TDJ6 403 Q8TDW7 404 Q8TEU7 405 Q8WUM4 406 Q8WVV7 407 Q8WVW5 408Q8WY27 409 Q92484 410 Q92820 411 Q92863 412 Q92905 413 Q92973 414 Q96AA1415 Q96HI4 416 Q96I12 417 Q96KR9 418 Q96LC1 419 Q96P44 420 Q96RY7 421Q96SM3 422 Q96ST3 423 Q99436 424 Q99598 425 Q99707 426 Q99832 427 Q9BT36428 Q9BT78 429 Q9BTY9 430 Q9BWV3 431 Q9BX63 432 Q9BXS5 433 Q9BXT5 434Q9C0C2 435 Q9H0G2 436 Q9H1W6 437 Q9H3E1 438 Q9H3Q7 439 Q9HC03 440 Q9NR71441 Q9NU86 442 Q9NWN9 443 Q9NYC9 444 Q9P0S0 445 Q9UCB0 446 Q9UCS3 447Q9UE54 448 Q9UF55 449 Q9UGA0 450 Q9UHV7 451 Q9UIW2 452 Q9UKP4 453 Q9UKU9454 Q9ULC4 455 Q9ULJ3 456 Q9UNA2 457 Q9UNM6 458 Q9UPV8 459 Q9Y266 460S4R3N1 461 S4R3S7 462 S4R400 463 T1S9D5 464 V9HWF8 465 W4VSQ3 466 W8SJH0467 X6R6S3

TABLE 6 Proteins in NPEX and APEX 1 A0A024R3G7 2 A0A024R8E2 3 A0A087WTU94 A0A087X191 5 A0A0A0MSS2 6 A0A0A0MTL9 7 A0A0A6YYJ5 8 A0A0J9YY72 9A4FTX9 10 A5D906 11 A5HML1 12 B4DDH4 13 B4DDI8 14 B4DFH6 15 B4DJQ7 16B4DV85 17 B4DXB2 18 B7Z7R2 19 B7Z8P5 20 B3KSE9 21 B4DIA1 22 B4DMS3 23B4E2P9 24 B4E315 25 B7Z380 26 B7Z403 27 B7Z625 28 B7Z8W6 29 B7ZLP5 30C9J2C0 31 C9JER5 32 E7EUT5 33 E9PFH7 34 E9PKG1 35 G1UI17 36 H0Y871 37H0YME5 38 H3BMM5 39 H9KV48 40 J3QS51 41 K7EIJ8 42 K7ELF8 43 K7ELK0 44A0A140CTX8 45 A8K3Y5 46 B4DFM5 47 B4DGH0 48 B4DWX3 49 B4DXM1 50 B7Z78051 C9JMY1 52 A0A024R9W5 53 A0A068F7M9 54 A0A0A0MQZ2 55 A0A0K0Q2Z1 56A8K719 57 A8KAC4 58 B2R6V9 59 B3KW36 60 B4DEN5 61 B4DJX6 62 B4DMD5 63B4DMN1 64 B4DPD5 65 B4DUJ5 66 B4DZR3 67 B7ZLC9 68 B7ZLX9 69 F5H6J0 70F8VV04 71 H0YEZ5 72 H7C1I7 73 J3KS22 74 K7ESP4 75 A0A126GW47 76 B3KVV677 B4DEQ2 78 B4DL70 79 B4E3H4 80 B9EK46 81 C9JJE5 82 C9K0S6 83 A8K7H3 84A9UEZ6 85 B3KNM0 86 D4YW74 87 F8W888 88 G3V200 89 G3V3A7 90 H3BT58 91A0A024RAM0 92 A0A0G2JS52 93 A0A0U1ZI18 94 A8K972 95 A8K9U4 96 A8KA50 97B2R6U8 98 B3KMK2 99 B4DV51 100 B4DVV1 101 B4DYQ4 102 B4DZP8 103 B5BUC0104 B7Z1E1 105 B7Z7U9 106 A0A024RA21 107 A0A087X201 108 A2MYD1 109A8K6R5 110 A8K919 111 B2R7E8 112 B3KXS5 113 B4DIX9 114 C5J0G2 115 C9J5X1116 E9PGN7 117 F5H7S7 118 H7BY57 119 I3L2J8 120 J3KR44 121 A0A140VK43122 A8K2P6 123 B7Z6W9 124 A0A087WXD4 125 B3KT18 126 A0A024R333 127A0A024R637 128 A0A059U7G5 129 A0A0C4DGM5 130 A0A0S2Z4F3 131 A6NHN2 132A8K291 133 B2R4S5 134 B4DDN3 135 B4DDS9 136 B4DFK1 137 B4DM68 138 B4DNJ2139 B4DRC6 140 B4DZV8 141 B4DZW2 142 B5A928 143 D3DWL9 144 E5RFX4 145E7ENU7 146 F8WBE0 147 H0YCC0 148 H7BYM0 149 I3NI20 150 J3KP74 151 J3KRC3152 O60391 153 O60486 154 O60760 155 O75322 156 O76061 157 P02549 158P11216 159 P20908 160 P27105 161 P42694 162 P42702 163 P48426 164 P54578165 P59998 166 P60660 167 P61812 168 Q01995 169 Q0VAP0 170 Q14571 171Q15181 172 Q15670 173 Q1WWK5 174 Q1WWK6 175 Q4LE40 176 Q53EU0 177 Q53GP3178 Q53H16 179 Q562U0 180 Q580X3 181 Q59EA4 182 Q59ES1 183 Q59EY7 184Q59G44 185 Q59G78 186 Q59GJ6 187 Q5GIA7 188 Q5H9N4 189 Q5T765 190 Q5TDF0191 Q5VT06 192 Q5W0B7 193 Q5XJ04 194 Q61N74 195 Q6NSB2 196 F8W9Z1 197H0YHD9 198 F6TR53 199 H0Y796 200 A7E2C0 201 B0I1R1 202 A0A0G2JNC5 203B3KNW3 204 B4DGV1 205 D6RJD3 206 E9PMI5 207 H0YD65 208 O43491 209 O95782210 P16035 211 P19320 212 Q05D08 213 Q12771 214 Q2QD09 215 Q2VPJ6 216Q59HC8 217 Q5R210 218 Q5VZB5 219 Q68D26 220 A0A024R566 221 A0A024R899222 A0A087WTK0 223 A0A087WWY8 224 A0A0K0K1L8 225 A0A0U1RRM0 226 A0JLR0227 A4F4K4 228 A4FVC3 229 A6NLU0 230 A8K1G1 231 B4DEX9 232 B4DF07 233B4DHL2 234 B4DJ31 235 B4DRK5 236 B7Z6W5 237 B7ZAG8 238 C9JCJ5 239 C9JQU8240 E5RIA9 241 E9PI86 242 F5H5K0 243 F8W1A4 244 H0Y831 245 H0YF06 246H0YLN7 247 H3BRP2 248 H7BYJ9 249 J3KR05 250 J3KTP9 251 K7EJ78 252 A6MW40253 A6NIW5 254 B3KM58 255 B3KXP1 256 B4DFC4 257 B4DS57 258 B4DVN5 259B7Z670 260 J3QSF4 261 O43242 262 O75554 263 P02771 264 P07476 265 P13521266 P52951 267 Q53FB6 268 Q53G25 269 Q53GZ5 270 Q53H60 271 Q53HR1 272Q53SW3 273 Q59FX3 274 Q59GW6 275 Q59H85 276 Q5GJ68 277 Q5U0Q1 278 Q5VWT9279 Q68DF0 280 Q6ICB4 281 A0A0S2Z455 282 B4DHJ3 283 B4E354 284 B5BTY4285 O75952 286 A0A024R5W0 287 A8K607 288 B3KTA8 289 Q16798 290 B7Z7A4291 G3V504 292 H0YCA1 293 B3KS69 294 F8W062 295 A0A024QZ45 296A0A0A0MTE8 297 A0A0A1TTP8 298 A4FU65 299 A8K8J2 300 A8K9A5 301 B3KQQ7302 B4DI59 303 B4DNH7 304 B4DVM1 305 B4E2K5 306 B4E3M8 307 B7Z6H0 308B8XXQ3 309 D6RBD3 310 D6W632 311 E5RI06 312 E7EPB6 313 E7ERH2 314 E7ERU0315 E7ETU9 316 E9PMI6 317 F5GX94 318 F5GYF1 319 F8VRQ0 320 F8VRZ4 321G3V2J8 322 G3V595 323 H0YAN8 324 H0YCE8 325 H0YJK0 326 M0QXS6 327 P08697328 P61604 329 Q13415 330 Q1L857 331 Q4FEB4 332 Q53FV3 333 Q53G79 334Q5SPU2 335 Q6IS14 336 P29317 337 B4DHN0 338 A0A024QZL4 339 A0A024R235340 A0A024R8H6 341 A0A087WZX0 342 A0A0G2JNJ8 343 B4DFL3 344 B4DVM7 345I6L9I5 346 P84243 347 Q0EFC9 348 Q13787 349 Q15102 350 Q6PK50 351 Q6PKT5352 Q6UVJ0 353 Q6ZP82 354 Q7Z3P6 355 Q7Z5Q5 356 Q7Z5V0 357 Q7Z5V6 358Q86UF4 359 Q86Y65 360 Q8IY52 361 Q8IYA8 362 Q8IZ02 363 Q8N754 364 Q8N948365 Q8N959 366 Q8N995 367 Q8NEZ3 368 Q8NHP6 369 Q8NI77 370 Q8TA90 371Q8TCL2 372 Q8TE54 373 Q8WU08 374 Q8WXI3 375 Q8WY81 376 Q96AP5 377 Q96DF0378 Q96M29 379 Q9BZ26 380 Q9H197 381 Q9H3W5 382 Q9H4B7 383 Q9H5H4 384Q9H7K8 385 Q9H853 386 Q9NQY1 387 Q9NS29 388 Q9P019 389 Q9P2Y4 390 Q9UBV4391 Q9UEH4 392 Q9UHV4 393 Q9UKN7 394 Q9UKV8 395 Q9UKX3 396 Q9UNP4 397Q9UQH3 398 Q9UQM3 399 Q9Y2D4 400 Q9Y2I4 401 Q9Y2J7 402 Q9Y5T6 403 Q9Y617404 S4R2Z6 405 S4R3K3 406 S4R457 407 V5N4G2 408 V9HW13 409 V9HW39 410V9HW77 411 V9HWD6 412 X5D2V8 413 Q7Z407 414 Q86UP2 415 Q8N3Y0 416 Q8TB46417 Q8TBN9 418 Q92736 419 Q96QD5 420 Q99698 421 Q9H281 422 Q9HCU0 423Q9UC36 424 Q9UML6 425 X5D9D6 426 X6R3L3

TABLE 7 Proteins unique to MSCEX 1 A0A024QYT5 2 A0A024QZN8 3 A0A024R0594 A0A024R1C3 5 A0A024R241 6 A0A024R2M8 7 A0A024R407 8 A0A024R4B7 9A0A024R4W0 10 A0A024R5D9 11 A0A024R5G9 12 A0A024R5Z8 13 A0A024R6C0 14A0A024R6F8 15 A0A024R6I1 16 A0A024R6L8 17 A0A024R6X4 18 A0A024R8B3 19A0A024R8V7 20 A0A024R930 21 A0A024R9X6 22 A0A024RB85 23 A0A024RCP3 24A0A068B0Y9 25 A0A068BI38 26 A0A087WSZ1 27 A0A087WTU7 28 A0A087WW54 29A0A087WXB0 30 A0A087WY10 31 A0A087WZV0 32 A0A087X0V8 33 A0A088QF11 34A0A0A0MRF6 35 A0A0A0MRS7 36 A0A0A0MRZ9 37 A0A0A0MTE1 38 A0A0A1TE42 39A0A0A6YY96 40 A0A0B4J1S7 41 A0A0C4DFX6 42 A0A0C4ZN31 43 A0A0G2JMD2 44A0A0G2JNH7 45 A0A0G2JNU3 46 A0A0G2JQU7 47 A0A0J9YY34 48 A0A0K0KS85 49A0A0S2Z3G9 50 A0A0S2Z645 51 A0A0U1RQC7 52 A0A0U1RRH9 53 A0A140F1N4 54A0A140T9S2 55 A0A140VK13 56 A0AVL2 57 A0PJ76 58 A0PJM7 59 A1KZ92 60A1L3V6 61 A2I2N5 62 A2RRC9 63 A2VDK1 64 A6NFQ2 65 A6NFU0 66 A7MAY2 67A8K0Z6 68 A8K2M5 69 A8K454 70 A8K5X8 71 A8K6R9 72 A8K9U6 73 A8MYV6 74A9YTQ3 75 B0QYK4 76 B1AHR3 77 B1AJW0 78 B1AUU8 79 B2R6H3 80 B2R6X6 81B2R6Y1 82 B2R702 83 B2R928 84 B2R969 85 B2RB72 86 B2RC85 87 B2RE36 88B2RWN6 89 B3KQ04 90 B3KR61 91 B3KRU1 92 B3KS75 93 B3KSD8 94 B3KTT6 95B3KU51 96 B3KUR8 97 B3KV69 98 B3KW35 99 B3KW47 100 B3KXX8 101 B4DDE5 102B4DE87 103 B4DER4 104 B4DEV8 105 B4DFA5 106 B4DFX9 107 B4DHR9 108 B4DIA7109 B4DIH7 110 B4DNL5 111 B4DNW7 112 B4DPQ0 113 B4DQI9 114 B4DQQ0 115B4DR29 116 B4DSP1 117 B4DST5 118 B4DTZ9 119 B4DU14 120 B4DWE2 121 B4DWG5122 B4DWQ4 123 B4DXC4 124 B4DYB8 125 B4DYX7 126 B4DZK1 127 B4DZP2 128B4E0I8 129 B4E164 130 B4E1Z4 131 B4E295 132 B4E3R6 133 B4E3S9 134 B5MCN7135 B7Z2D6 136 B7Z2E6 137 B7Z3I0 138 B7Z3K9 139 B7Z427 140 B7Z4H0 141B7Z549 142 B7Z5X3 143 B7Z8B9 144 B7Z9H7 145 B7Z9J0 146 B7ZAB8 147 B7ZAL5148 B7ZL25 149 B7ZMF3 150 B8ZZH7 151 C4B4C6 152 C9J4M6 153 C9J613 154C9JGT3 155 C9JGV6 156 C9JLB7 157 C9JQI2 158 C9JSI2 159 C9JT28 160 C9JYI9161 D2KTB5 162 D2XBF0 163 D3DV75 164 D3DVD8 165 D6R9U7 166 D6RA00 167D6RF53 168 D6RGY2 169 D6W633 170 E3W994 171 E5KMI6 172 E5KR05 173 E5KRK5174 E5KTI5 175 E5RGN8 176 E5RHI0 177 E7ENN3 178 E7EUY3 179 E7EWC2 180E7EX20 181 E9PKT1 182 E9PQI5 183 E9PR16 184 F2Z2C8 185 F2Z2K5 186 F5GX59187 F5GYJ8 188 F5H065 189 F5H226 190 F5H432 191 F5H4B6 192 F6TLX2 193F8VU88 194 F8VXG7 195 F8VXL3 196 F8VY04 197 F8W0U9 198 F8W9U4 199 F8W9W0200 F8WCF6 201 G1UI33 202 G5E971 203 G5ELZ6 204 H0Y468 205 H0Y5R6 206H0Y6I0 207 H0Y8G5 208 H0YAN3 209 H0YBI2 210 H0YC83 211 H0YCJ2 212 H0YEL4213 H0YH65 214 H0YL91 215 H0YM70 216 H3BP20 217 H3BSC0 218 H3BSE1 219H3BSM2 220 H3BTW3 221 H3BV41 222 H7BYJ1 223 H7BYX7 224 H7C074 225 H7C1W8226 I3L3E8 227 I3L3P7 228 J3KN38 229 J3KNC6 230 J3KPK1 231 J3KQ09 232J3KTB8 233 J3QL70 234 K0I859 235 K7EIS7 236 K7EKQ3 237 K7EL65 238 K7ELC2239 K7ELW0 240 K7EMJ5 241 K7EMR1 242 K7EPJ9 243 K7ESE0 244 L0R6V3 245L8E853 246 M0R1K3 247 M0R361 248 O00469 249 O00562 250 O00592 251 O14530252 O14879 253 O15013 254 O15042 255 O15131 256 O43505 257 O43820 258O60245 259 O60266 260 O60294 261 O60296 262 O60343 263 O60522 264 O60543265 O60603 266 O75161 267 O75339 268 O75390 269 O75578 270 O94844 271O94854 272 O94925 273 O95165 278 P00519 279 P02538 280 P02786 281 P04054282 P04075 283 P04818 284 P05091 285 P05231 286 P05937 287 P06493 288P09622 289 P09960 290 P0DMV8 291 P10398 292 P13667 293 P13807 294 P15311295 P18065 296 P22670 297 P23109 298 P28065 299 P28482 300 P29074 301P30532 302 P31327 303 P34932 304 P35916 305 P40189 306 P42898 307 P46782308 P47755 309 P48058 310 P48643 311 P49863 312 P50748 313 P51828 314P61978 315 P62701 316 P63208 317 P68104 318 P78477 319 Q10570 320 Q10713321 Q12770 322 Q12905 323 Q12913 324 Q13241 325 Q13315 326 Q13765 327Q13885 328 Q14019 329 Q14112 330 Q14584 331 Q14678 332 Q14691 333 Q14980334 Q149N5 335 Q14CN4 336 Q15274 337 Q15431 338 Q15751 339 Q15878 340Q1L838 341 Q1RMC9 342 Q1W6H9 343 Q20BJ8 344 Q21096 345 Q2TA76 346 Q38SD2347 Q3KP44 348 Q3KRB0 349 Q3ZCN5 350 Q3ZCT4 351 Q495V9 352 Q4G0L2 353Q4G192 354 Q4QRK8 355 Q4VNC1 356 Q4W5F5 357 Q53GQ2 358 Q53HH4 359 Q53QE9360 Q53Y51 361 Q58P21 362 Q59E87 363 Q59G22 364 Q59G34 365 Q59GT7 366Q59GW5 367 Q59GX6 368 Q59HG1 369 Q5CCK6 370 Q5EBN2 371 Q5F0I5 372 Q5I0G3373 Q5J9B1 374 Q5NV87 375 Q5STU8 376 Q5SVJ3 377 Q5T764 378 Q5TEJ7 379Q5U608 380 Q5VU34 381 Q5VUJ6 382 Q5VYI1 383 Q5VYS4 384 Q5VZ00 385 Q5VZB4386 Q5VZL5 387 Q68CZ1 388 Q68DA4 389 Q68DB7 390 Q6EKJ0 391 Q6FI27 392Q6NT96 393 Q6P0N9 394 Q6P1K8 395 Q6P2S1 396 Q6P4R9 397 Q6P6D5 398 Q6PD62399 Q6UWZ7 400 Q6UXN9 401 Q70IA8 402 Q71U36 403 Q75MN6 404 Q7L6B3 405Q7Z3Z1 406 Q7Z4W1 407 Q7Z6M0 408 Q86T35 409 Q86U18 410 Q86UU0 411 Q86UW9412 Q86V60 413 Q86XL3 414 Q86YL5 415 Q8IVH2 416 Q8IWP9 417 Q8IYA6 418Q8N304 419 Q8N3P5 420 Q8N4P6 421 Q8N5U0 422 Q8N7F5 423 Q8NBB8 424 Q8NE35425 Q8NES3 426 Q8NFP9 427 Q8NFQ8 428 Q8NGS8 429 Q8TAA3 430 Q8TC08 431Q8TEQ6 432 Q8WVC6 433 Q8WVJ9 434 Q8WWJ6 435 Q8WWL7 436 Q8WXI9 437 Q8WYG9438 Q8WYJ0 439 Q92530 440 Q92539 441 Q92823 442 Q969U7 443 Q96A69 444Q96B09 445 Q96B65 446 Q96CU9 447 Q96CZ8 448 Q96F45 449 Q96FJ0 450 Q96GE4451 Q96HY6 452 Q96JB2 453 Q96JM4 454 Q96MM8 455 Q96N57 456 Q96PX1 457Q96QF4 458 Q96QI5 459 Q96S69 460 Q99539 461 Q99574 462 Q9BPU6 463 Q9BQN1464 Q9BQS2 465 Q9BS61 466 Q9BSD7 467 Q9BW71 468 Q9BWD1 469 Q9BWT3 470Q9BXB5 471 Q9BXT8 472 Q9BYF1 473 Q9C091 474 Q9H0Q0 475 Q9H2E5 476 Q9H2I8477 Q9H2Z3 478 Q9H7U7 479 Q9H7W5 480 Q9H8N6 481 Q9H8Q1 482 Q9HA17 483Q9HAR0 484 Q9HD74 485 Q9NP17 486 Q9NPU7 487 Q9NR45 488 Q9NRD1 489 Q9NT26490 Q9NTG1 491 Q9NW02 492 Q9NX58 493 Q9NX91 494 Q9NZF5 495 Q9P283 496Q9P291 497 Q9P2M7 498 Q9P2N1 499 Q9UBG9 500 Q9UBL0 501 Q9UC78 502 Q9UEH6503 Q9UF08 504 Q9UFA7 505 Q9UIA9 506 Q9UJD0 507 Q9UJT0 508 Q9UL70 509Q9ULJ7 510 Q9ULW6 511 Q9UME3 512 Q9UNM1 513 Q9UNS1 514 Q9UPN4 515 Q9UPV0516 Q9Y216 517 Q9Y4L1 518 Q9Y613 519 Q9Y696 520 Q9Y6K1 521 Q9Y6S7 522Q9Y6U9 523 R4GMT0 524 S4R2X0 525 S4R314 526 S4R3C8 527 U3KQK0 528 U5YBI9529 V9GY77 530 V9GYE3 531 V9HW26 532 V9HWC4 533 V9HWH0 534 X5D7Q2 535X5D9D2 536 X6RL08

TABLE 8 Proteins unique to APEX 1 A0A087WXN9 2 A0A0A0MTD1 3 A0A0C4DFS8 4A0A0J9YX41 5 A0A140VJI6 6 A2VED2 7 B0YJ74 8 A0A0B4J1X7 9 A0A5A9 10A8K1P5 11 B3KMN2 12 B5A934 13 B7Z9C0 14 A0A087X1U1 15 A8K0G7 16 B7ZLC417 A0A140VKF7 18 B4DY95 19 A0A024R1K7 20 A0A024RCS3 21 A0A0G2JPN1 22A0A140VJP0 23 B3KR94 24 B3KSB6 25 B4DET1 26 B4DG15 27 B4DJI2 28 B7Z91929 C9J3L8 30 A0A087X298 31 B4DG65 32 A0A0C4DH13 33 B4DSP4 34 B4DSR4 35C9J826 36 A0A024RBC0 37 A0A087X120 38 A0A0C4DGC5 39 A0A0U1WUY1 40 A1L19641 A9CQZ2 42 B3KX52 43 A0A087X0D8 44 B2RCJ5 45 B4DN90 46 B4DNZ9 47B4DZG1 48 B7Z6I1 49 C9J1D9 50 C9J1K7 51 A0AV47 52 B3KNC3 53 A0A024R12054 A0A087WUL9 55 B3KMX4 56 B3KNL6 57 B3KPZ7 58 B3KWB6 59 B3KWC4 60D1CS35 61 D2CPK5 62 D3DUJ0 63 D6REL5 64 E5RHP0 65 E9PCP0 66 E9PJL7 67E9PNX2 68 F5H0Y9 69 F6QTA4 70 F8W026 71 G3V5R6 72 G5E9J0 73 H0Y757 74H0Y9N5 75 H0YDE4 76 H0YGV8 77 H3BTQ9 78 H7BZZ8 79 H7C0D9 80 J3KPD9 81J3KTL9 82 K7EL58 83 K7EMM9 84 K7EQR9 85 A2RRR7 86 B4DFQ9 87 B4DWW6 88A8MT18 89 A0A024R461 90 B4DZ23 91 B7ZMJ6 92 E5RJD0 93 I7HAS0 94A0A075B610 95 A0A087WU14 96 A0A0U1ZBR1 97 A0A140TA43 98 A0A140VK41 99B2RA91 100 B3KNB4 101 B4DMA2 102 B4DTA5 103 B4DUF8 104 A0A0C4DGH5 105A6NEP9 106 A8K0K0 107 B3KMS6 108 B3KY88 109 B7Z4X0 110 E7ETB4 111 E9PGC0112 F5H0M4 113 F5H5D3 114 H0Y463 115 H7C080 116 I3L0G6 117 I3L1K1 118K7EJX8 119 K7EK57 120 A0A024R283 121 A0A0C4DGB8 122 A0A140VK27 123B4DY04 124 B7ZM05 125 K7ENN8 126 B2R8P5 127 H0Y873 128 A0A024R542 129A0A024R7E1 130 A0A0M4FNU3 131 B4DUC5 132 B4DZ03 133 A0A024R957 134A0A0A0MRY9 135 A0A140VK76 136 B0YJ32 137 B3KPK8 138 B4DEY4 139 B4DMJ7140 B4DNX8 141 B4DQY2 142 B4DR52 143 B4DR67 144 B4DTA9 145 B4E058 146B7ZAW7 147 B7ZLB7 148 E9PNS9 149 H7BXG6 150 H7C520 151 I3L466 152A0A0A0MRM2 153 A4FU77 154 A0A024QZ75 155 A0A024R674 156 A0A0A0MRH0 157A8K4M1 158 B3KTQ2 159 B4DJ21 160 B4DMJ5 161 B4DV55 162 D3DSV6 163 D6R9D5164 E9PP50 165 H0Y2M6 166 H0YCV9 167 H0YJ91 168 H7C5H1 169 J3QRV6 170L8E9P0 171 M0R120 172 O00512 173 O00763 174 O14524 175 O15078 176 O15090177 O15244 178 O43570 179 O60291 180 O75094 181 O75600 182 P00352 183P02749 184 P05546 185 P08910 186 P10916 187 P17927 188 P27169 189 P29400190 P35499 191 P35749 192 P39023 193 P40939 194 P46059 195 P50225 196P54756 197 P61964 198 P84085 199 Q003V9 200 Q01831 201 Q0VAI6 202 Q12873203 Q12980 204 Q13349 205 Q14145 206 Q14469 207 Q15238 208 Q15697 209Q15762 210 Q16820 211 Q2TSD0 212 Q32Q67 213 Q3SY52 214 Q53G92 215 Q53GF5216 Q53HR5 217 Q53SE2 218 Q58DX5 219 Q59EF3 220 Q59HA9 221 Q5GJ67 222Q5TEU4 223 Q68DP4 224 Q69YH5 225 Q6FHK0 226 Q6FHK9 227 Q6FHU3 228 Q6FI03229 Q6IFE0 230 Q6JH03 231 Q6NUI8 232 O15123 233 O43301 234 O43424 235O95239 236 P02533 237 P55283 238 Q15142 239 Q1HP67 240 Q562N6 241 Q5H9B5242 Q5HY54 243 Q5HYL7 244 Q5VUA0 245 Q6IAX6 246 Q6P1L4 247 Q6P5Q4 248B3KXC1 249 B3KP29 250 B7Z7C2 251 A0A024RDT3 252 A8K990 253 E5RGT9 254E9PMW9 255 G3V174 256 H7BXS7 257 H7C402 258 H9A532 259 H3BP35 260A0A024RDR3 261 B4DEW9 262 C9J3M5 263 I0CMK4 264 K7ENA0 265 B3KNV9 266B4DUK7 267 K7EPF6 268 A0A024QZF7 269 A0A024R5V2 270 M0QY71 271 O75843272 O95905 273 O96011 274 P07585 275 Q0VD99 276 Q15374 277 Q5VX85 278Q61C98 279 P30405 280 Q5VIR6 281 A0A0C4DFQ8 282 B1AMW3 283 A0A087WUF5284 A0A024R6K8 285 B2R9P0 286 A0A087WSV7 287 A0A0K0KR34 288 A6NFY4 289A0A024RD47 290 B2RTX2 291 E9PFF2 292 F2Z2G2 293 F5GWA7 294 F5GXC7 295F5GYF8 296 F5H3Q5 297 H9C875 298 B7Z2E2 299 J3KQ72 300 A0A024R909 301F5GXR6 302 J3KS93 303 A0A126GVW7 304 B4E266 305 A0A024R2H7 306A0A097CK88 307 A0A140T999 308 A8K8H7 309 B4DND2 310 B4DQM1 311 B4DZ35312 E7ERI8 313 F5GZQ4 314 F8WFC4 315 H0Y9E9 316 H7C1T2 317 K7EPY4 318M0R2D3 319 O76074 320 P15085 321 Q3YA63 322 Q5MNZ6 323 Q6IPX8 324A0A087X2E5 325 A0A0A0MQR7 326 A0A0C4DGY8 327 A0A0D9SF28 328 A0A0S2Z333329 A0A0S2Z366 330 A4D202 331 A8K731 332 B3KX01 333 B3KX41 334 B4DEM5335 B4DK22 336 B4E337 337 B7Z4N1 338 C9J8U1 339 D6R9E4 340 E7EP61 341E7EWZ1 342 E9KL23 343 E9PJK2 344 F5GXY2 345 F5H032 346 G3V454 347 H0YF19348 H0YGH1 349 H3BRL9 350 K0A7K7 351 A0A0G2JN61 352 A0A0M4FEM1 353A0A0U1RQL8 354 A8K6X5 355 B3KW51 356 B4DQG4 357 B4DW50 358 B4DZY4 359B4E1S5 360 B4E1T9 361 B4E3A4 362 B7Z1Y2 363 C9JUE0 364 D6RDI0 365 F8VUX9366 J9UPX7 367 K7ENE1 368 P13637 369 P28827 370 P31150 371 P31946 372Q12769 373 Q13841 374 Q20BG7 375 Q2F838 376 Q58FF6 377 Q658X2 378 Q659A9379 Q6FGX9 380 A0A024R3D4 381 A0A024RAM6 382 A0A0G2JP38 383 A0A140VJV9384 A0A140VKF6 385 B4DLH5 386 D6RHE5 387 O14777 388 Q6IBD2 389A0A024R684 390 A8K5F3 391 B3KU50 392 B4DUZ8 393 K7EQ48 394 O75665 395Q59EK9 396 A0A024R7D2 397 A3KFK1 398 G3V0E6 399 A0A024QYZ8 400A0A024RAM1 401 A0A087WWI0 402 A0A0A0MQR2 403 A4D0S8 404 A4FU51 405A4QMW8 406 A8K4Q4 407 C9JVG3 408 D6RC52 409 E7ENJ7 410 E9PHK0 411 E9PQV8412 F5GZI5 413 F5GZK2 414 F5H2D0 415 F5H6I7 416 F6U236 417 F8WAS2 418F8WAX8 419 F8WCJ1 420 H3BTW5 421 H3BUA3 422 K7EJH1 423 M5EDK8 424 O00160425 O15054 426 P48444 427 Q14258 428 Q14434 429 Q14690 430 Q549N0 431Q6FH62 432 A0A024RB92 433 A0A087WW76 434 B4DI80 435 B4DTQ1 436 B4E0B2437 D6RE83 438 D9HTE9 439 E9PMM8 440 E9PNE6 441 F2Z393 442 F5H0P4 443H0Y476 444 H0YCK3 445 J3KRJ6 446 J3QS44 447 O75077 448 P06727 449 P20062450 Q09472 451 Q14643 452 Q6PKD3 453 Q6QWC0 454 Q6RBX8 455 Q6UW88 456Q6UX71 457 Q6ZMU5 458 Q6ZP56 459 Q6ZPD6 460 Q6ZR36 461 Q6ZU35 462 Q6ZVM7463 Q709C8 464 Q7M4R4 465 Q7Z2V5 466 Q7Z606 467 Q7Z612 468 Q7Z668 469Q7Z7H5 470 Q7Z7P9 471 Q86SG7 472 Q86TE2 473 Q86TI0 474 Q86UE3 475 Q86X82476 Q86XM6 477 Q86XQ3 478 Q86YC4 479 Q8IUN7 480 Q8IUQ0 481 Q8IV08 482Q8IWJ6 483 Q8IXA4 484 Q8IYT1 485 Q8N1M4 486 Q8N1Y9 487 Q8N4M9 488 Q8NC18489 Q8NCS7 490 Q8NGA0 491 Q8TBG4 492 Q8TCC9 493 Q8WLP1 494 Q8WV67 495Q8WVV4 496 Q8WYB5 497 Q92574 498 Q92615 499 Q92932 500 Q92990 501 Q969V5502 Q96FW1 503 Q96G03 504 Q96HX7 505 Q96KR4 506 Q96MY1 507 Q99062 508Q99440 509 Q99973 510 Q99989 511 Q9BT21 512 Q9BT27 513 Q9BVJ8 514 Q9BWT2515 Q9BZF9 516 Q9C0A1 517 Q9H295 518 Q9H2M9 519 Q9H4N8 520 Q9H6A9 521Q9H6L3 522 Q9H6Z9 523 Q9H769 524 Q9H995 525 Q9HBM3 526 Q9NPL4 527 Q9NS28528 Q9NUJ7 529 Q9NWV8 530 Q9NX81 531 Q9NX98 532 Q9NXP1 533 Q9NYK1 534Q9NZ52 535 Q9P0C1 536 Q9P0P1 537 Q9P2H3 538 Q9UBK7 539 Q9UDV6 540 Q9UDX4541 Q9UFP2 542 Q9UFU2 543 Q9UG36 544 Q9UG64 545 Q9UGU8 546 Q9UH61 547Q9UHC1 548 Q9UHC9 549 Q9ULH1 550 Q9UNX4 551 Q9UPS8 552 Q9Y287 553 Q9Y4G6554 S4R3L5 555 T1WFC1 556 U3KPS2 557 U5YEI7 558 V9GYM3 559 V9HW42 560V9HWC2 561 V9HWC3 562 V9HWG1 563 W6JLH6 564 W8SBA0 565 Q6ZNY8 566 Q6ZP01567 Q7Z5U6 568 Q86TA0 569 Q86VI3 570 Q86WN1 571 Q86YG0 572 Q8IWK6 573Q8IXR5 574 Q8N111 575 Q8N976 576 Q8NG48 577 Q8TCU4 578 Q8WYK0 579 Q99994580 Q9BRL4 581 Q9BS12 582 Q9BTS8 583 Q9C0K3 584 Q9H583 585 Q9HBG7 586Q9P2H0 587 Q9UF83 588 Q9UHP3 589 Q9UIT6 590 Q9UJ65 591 Q9UN47 592 Q9UNY4593 U3KQU8 594 X5D5A9 595 X5D7Z6 596 X5DNR2

TABLE 9 Proteins unique to NPEX 1 A0A024DAK3 2 A0A024QYX2 3 A0A024QZJ1 4A0A024QZM2 5 A0A024QZW5 6 A0A024QZX5 7 A0A024R095 8 A0A024R0A0 9A0A024R233 10 A0A024R2G9 11 A0A024R2K1 12 A0A024R301 13 A0A024R4A0 14A0A024R4D1 15 A0A024R4F1 16 A0A024R4Z0 17 A0A024R4Z5 18 A0A024R5M9 19A0A024R5U3 20 A0A024R5V6 21 A0A024R5Y2 22 A0A024R6G3 23 A0A024R701 24A0A024R7B6 25 A0A024R7L5 26 A0A024R7T7 27 A0A024R884 28 A0A024R8F4 29A0A024R814 30 A0A024R936 31 A0A024R944 32 A0A024RAF7 33 A0A024RAS2 34A0A024RB01 35 A0A024RBE5 36 A0A024RBT9 37 A0A024RCB7 38 A0A024RCE1 39A0A024RCT0 40 A0A024RD04 41 A0A024RD09 42 A0A024RD36 43 A0A024RD77 44A0A024RDM3 45 A0A024RDV8 46 A0A024RDW5 47 A0A075B6S4 48 A0A075B734 49A0A087WSW7 50 A0A087WT58 51 A0A087WTH0 52 A0A087WTK9 53 A0A087WU78 54A0A087WUK8 55 A0A087WUM0 56 A0A087WVI0 57 A0A087WW59 58 A0A087WWE7 59A0A087WXK5 60 A0A087WXM9 61 A0A087WXX8 62 A0A087WYC6 63 A0A087WYK9 64A0A087WYW3 65 A0A087WZ40 66 A0A087WZC4 67 A0A087WZR8 68 A0A087WZZ7 69A0A087X052 70 A0A087X0D6 71 A0A087X176 72 A0A087X1Z6 73 A0A087X250 74A0A087X2B0 75 A0A090N8H8 76 A0A097IQZ3 77 A0A0A0MQS0 78 A0A0A0MRP0 79A0A0A0MRT2 80 A0A0A0MRV0 81 A0A0A0MRX4 82 A0A0A0MS45 83 A0A0A0MS53 84A0A0A0MS59 85 A0A0A0MS84 86 A0A0A0MSA7 87 A0A0A0MSU4 88 A0A0A0MT74 89A0A0A0MTC4 90 A0A0A0MTD9 91 A0A0A0MTQ1 92 A0A0A0MTQ8 93 A0A0A0N0L2 94A0A0A0N0M1 95 A0A0B4J1R2 96 A0A0B4J210 97 A0A0B4J212 98 A0A0B4J223 99A0A0B4J2C3 100 A0A0C4DFX7 101 A0A0C4DG17 102 A0A0C4DG82 103 A0A0C4DGA6104 A0A0C4DGI2 105 A0A0C7DW92 106 A0A0D9SF54 107 A0A0D9SF63 108A0A0D9SG17 109 A0A0E3JG42 110 A0A0G2JHI3 111 A0A0G2JI86 112 A0A0G2JM47113 A0A0G2JNH0 114 A0A0G2JP14 115 A0A0G2JP37 116 A0A0G2JRM9 117A0A0G2JRN3 118 A0A0J9YVY3 119 A0A0J9YVZ3 120 A0A0J9YWL0 121 A0A0J9YWY2122 A0A0K1JS24 123 A0A0K2GMW5 124 A0A0S2Z3P7 125 A0A0S2Z451 126A0A0S2Z489 127 A0A0S2Z4F1 128 A0A0S2Z4H6 129 A0A0S2Z419 130 A0A0S2Z4R1131 A0A0S2Z4Y4 132 A0A0S2Z4Y6 133 A0A0S2Z563 134 A0A0S2Z5B0 135A0A0S2Z5Z7 136 A0A0S2Z618 137 A0A0U1RQH4 138 A0A0U1RQP0 139 A0A0U1RQR9140 A0A0U1RRM1 141 A0A126GVG1 142 A0A126GWA2 143 A0A126LAY8 144A0A126LB32 145 A0A140HDC1 146 A0A140T8X2 147 A0A140T9Y3 148 A0A140TA40149 A0A140TA77 150 A0A140VJC9 151 A0A140VJI0 152 A0A140VJL3 153A0A140VJN8 154 A0A140VJP5 155 A0A140VJU3 156 A0A140VK35 157 A0A141PNN4158 A0A146IHP0 159 A0A158T700 160 A0JLR2 161 A0PJY9 162 A1A512 163A1L3A3 164 A2A2M0 165 A2A368 166 A2ADX3 167 A2NX49 168 A2PYH4 169 A2RUH7170 A2VCK2 171 A4D126 172 A4D1B7 173 A4D1P7 174 A4D1R1 175 A4FU69 176A4FUA2 177 A4QPE5 178 A6H8W6 179 A6NC48 180 A6NC78 181 A6NED2 182 A6NF31183 A6NGH7 184 A6NGQ3 185 A6NHN7 186 A6NHT5 187 A6NM62 188 A6NN40 189A6NP61 190 A6QRJ1 191 A6XAA7 192 A7E294 193 A7E2X7 194 A7J1R0 195 A8E631196 A8K0E1 197 A8K146 198 A8K1W3 199 A8K3A3 200 A8K3L7 201 A8K3X2 202A8K4F0 203 A8K4I1 204 A8K525 205 A8K594 206 A8K5E6 207 A8K5H7 208 A8K5J8209 A8K6K4 210 A8K6V3 211 A8K7K0 212 A8K855 213 A8K8K1 214 A8K8T9 215A8MX12 216 A9QM74 217 A9UK01 218 B0QY51 219 B0QY53 220 B0QYP5 221 B1AKL4222 B1AKN6 223 B lAKN8 224 B1ALU6 225 B1B5Q8 226 B2R5U7 227 B2R694 228B2R6V2 229 B2R6X2 230 B2R734 231 B2R736 232 B2R7D2 233 B2R7I3 234 B2R7S8235 B2R7W6 236 B2R7Z4 237 B2R892 238 B2R9P8 239 B2R9S6 240 B2RA29 241B2RAN2 242 B2RB27 243 B2RC06 244 B2RCB8 245 B2RCD2 246 B2RCG9 247 B2RD40248 B2RDD7 249 B2RDT9 250 B2RNB2 251 B2RNT9 252 B2RUU1 253 B3FR89 254B3KM41 255 B3KM42 256 B3KMB1 257 B3KMB8 258 B3KMD2 259 B3KMJ7 260 B3KMZ6261 B3KN57 262 B3KP18 263 B3KPA6 264 B3KPD7 265 B3KPK9 266 B3KPM6 267B3KQ65 268 B3KQZ8 269 B3KR52 270 B3KRY3 271 B3KS09 272 B3KS20 273 B3KS48274 B3KS82 275 B3KSV0 276 B3KSW4 277 B3KSZ3 278 B3KTD8 279 B3KTM6 280B3KTR4 281 B3KU97 282 B3KUG5 283 B3KUL9 284 B3KUN1 285 B3KUR9 286 B3KUU1287 B3KV11 288 B3KVH9 289 B3KVJ1 290 B3KVN4 291 B3KVP2 292 B3KVU9 293B3KW07 294 B3KW31 295 B3KW52 296 B3KWI5 297 B3KWS8 298 B3KX72 299 B3KXH9300 B3KXW5 301 B3KXX5 302 B3KY97 303 B4DE05 304 B4DE48 305 B4DE80 306B4DEI6 307 B4DEX7 308 B4DF38 309 B4DFB6 310 B4DFF1 311 B4DFN8 312 B4DG42313 B4DGF4 314 B4DHN5 315 B4DHX3 316 B4DI94 317 B4DIB9 318 B4DIC2 319B4DJ53 320 B4DKC2 321 B4DKX2 322 B4DL63 323 B4DLP0 324 B4DM77 325 B4DMQ1326 B4DND4 327 B4DP10 328 B4DP52 329 B4DQ24 330 B4DQ80 331 B4DQD4 332B4DRG0 333 B4DRG2 334 B4DRT4 335 B4DS66 336 B4DSA0 337 B4DSC7 338 B4DSC8339 B4DT06 340 B4DTQ9 341 B4DUB2 342 B4DUY8 343 B4DV73 344 B4DVA7 345B4DWE9 346 B4DWF9 347 B4DWY2 348 B4DWZ8 349 B4DX03 350 B4DXP2 351 B4DXR7352 B4DXR8 353 B4DY09 354 B4DYD2 355 B4DYE6 356 B4DZ04 357 B4DZ99 358B4DZC2 359 B4DZC3 360 B4DZC9 361 B4DZD2 362 B4DZS8 363 B4E0B7 364 B4E0E1365 B4E0J9 366 B4E0Q4 367 B4E143 368 B4E173 369 B4E282 370 B4E2E2 371B4E2F7 372 B4E2W8 373 B4E2X3 374 B4E3A8 375 B4E3M6 376 B5A954 377 B5BU72378 B5MBY4 379 B5TYJ1 380 B7Z1G4 381 B7Z282 382 B7Z2F7 383 B7Z2X2 384B7Z2Z2 385 B7Z321 386 B7Z3A3 387 B7Z5H2 388 B7Z5N6 389 B7Z6E2 390 B7Z6K2391 B7Z6P1 392 B7Z6R5 393 B7Z6T0 394 B7Z6X5 395 B7Z7R8 396 B7Z7W2 397B7Z942 398 B7Z970 399 B7Z971 400 B7Z9C6 401 B7ZAR1 402 B7ZAV2 403 B7ZB07404 B7ZB16 405 B7ZBD5 406 B7ZBH1 407 B7ZL21 408 B7ZLC8 409 B7ZMI2 410B7ZMM1 411 B8ZZA5 412 B9A6J2 413 B9EG68 414 B9EG95 415 B9TWZ8 416 C4IXU6417 C6ZGQ9 418 C7S316 419 C9J0Q5 420 C9J268 421 C9J408 422 C9J524 423C9J712 424 C9J7G0 425 C9J8V3 426 C9JBE8 427 C9JCQ9 428 C9JEN8 429 C9JH19430 C9JSK8 431 C9JYQ2 432 D3DPF8 433 D3DS02 434 D3DSB5 435 D3DVJ3 436D3DVS8 437 D3DVT0 438 D3DXI8 439 D3DXI9 440 D3TTY9 441 D4IH22 442 D5KMU6443 D6R921 444 D6R936 445 D6R938 446 D6R9C2 447 D6R9E3 448 D6R9V7 449D6R9W4 450 D6RB55 451 D6RC76 452 D6RD46 453 D6RD74 454 D6RE68 455 D6RFH3456 D6RGK7 457 D6RIC7 458 D6RTK6 459 D7NTU0 460 E0Z3H0 461 E2RYF6 462E5RFZ0 463 E5RG94 464 E5RHK2 465 E5RJ52 466 E6Y365 467 E6Y3F9 468 E7EME3469 E7EMS9 470 E7ENT8 471 E7EP60 472 E7EPN9 473 E7ERS3 474 E7ESK9 475E7ET15 476 E7ETR9 477 E7EU35 478 E7EUC7 479 E7EV71 480 E7EV99 481 E7EVX8482 E9PB18 483 E9PB90 484 E9PCY5 485 E9PDF5 486 E9PEW0 487 E9PF18 488E9PG59 489 E9PGK7 490 E9PGW9 491 E9PIT3 492 E9PK85 493 E9PL71 494 E9PLE2495 E9PLF1 496 E9PMF9 497 E9PMH3 498 E9PMP7 499 E9PNL2 500 E9PPT0 501E9PQ63 502 E9PQ78 503 E9PQR7 504 E9PS38 505 E9PSI1 506 F1D8R5 507 F2FB34508 F2Z2C9 509 F2Z2V2 510 F5GXE6 511 F5GXV7 512 F5GY90 513 F5GYI5 514F5H081 515 F5H0Q0 516 F5H1B0 517 F5H248 518 F5H269 519 F5H2B9 520 F5H3F0521 F5H4R4 522 F5H5L5 523 F5H5P4 524 F5H6T1 525 F5H7H1 526 F6SS63 527F8LFZ0 528 F8VPW7 529 F8VS27 530 F8VXU5 531 F8VZR8 532 F8W0I7 533 F8W0P6534 F8W1Y6 535 F8W6X9 536 F8W8W4 537 F8WBD4 538 F8WEE2 539 G1UD80 540G2XKQ0 541 G3V119 542 G3V1R5 543 G3V2T5 544 G3V3H3 545 G3V5A2 546 G3V5B2547 G4V217 548 G5E9U2 549 G5E9W5 550 G5E9X1 551 G8JLD5 552 G9HZ40 553G9LIE3 554 H0UI21 555 H0Y2R3 556 H0Y494 557 H0Y5G0 558 H0Y626 559 H0Y6T7560 H0Y7U8 561 H0Y8A5 562 H0Y8J2 563 H0YA41 564 H0YA82 565 H0YAN2 566H0YC24 567 H0YC74 568 H0YDG2 569 H0YDS4 570 H0YE28 571 H0YGQ5 572 H0YGS7573 H0YI92 574 H0YIC9 575 H0YJN7 576 H0YKE9 577 H0YKJ0 578 H0YKU1 579H0YL26 580 H0YLC7 581 H0YMT1 582 H0YNC2 583 H3BMQ0 584 H3BMX7 585 H3BN32586 H3BNJ0 587 H3BPD5 588 H3BPM0 589 H3BQH0 590 H3BRR1 591 H3BRT3 592H3BRW2 593 H3BS46 594 H3BSK8 595 H3BT65 596 H3BTA7 597 H3BTB7 598 H3BTQ7599 H3BTZ4 600 H3BUN2 601 H3BUQ6 602 H3BV12 603 H6WCP5 604 H7BXH2 605H7BY59 606 H7BYG4 607 H7BZR8 608 H7BZR9 609 H7BZS7 610 H7C0N2 611 H7C150612 H7C276 613 H7C2M1 614 H7C2Q0 615 H7C2T4 616 H7C2Y1 617 H7C354 618H7C3W2 619 H7C440 620 H7C4T1 621 H7C525 622 H7C5Q0 623 H9C3J9 624 H9PCV7625 I1VYG6 626 I3L0A5 627 I3L194 628 I3L1M7 629 I3L305 630 I3L3Z0 631I6L9F7 632 I6T4L2 633 J3KP02 634 J3KQA9 635 J3KQJ9 636 J3KR49 637 J3KRL2638 J3KRL5 639 J3KS17 640 J3KS19 641 J3KSU0 642 J3KSZ0 643 J3KTE6 644J3KTF8 645 J3KTH2 646 J3QLL3 647 J3QQW1 648 J3QQX3 649 J3QS13 650 J3QSU0651 J3QT83 652 J7M5B9 653 K0IIP4 654 K7EID4 655 K7EIY4 656 K7EJZ2 657K7EL35 658 K7ELW4 659 K7EM44 660 K7EMC6 661 K7EMK6 662 K7ENP1 663 K7EQK2664 K7EQR0 665 K7ERD4 666 K7ERV2 667 K7ES09 668 K7ESB7 669 M0QXU3 670M0QZB5 671 M0R165 672 M0R3B5 673 M0R312 674 M1V481 675 M4QHP2 676 O00299677 O00363 678 O00443 679 O00444 680 O00451 681 O00462 682 O00471 683O00505 684 O00571 685 O00754 686 O14579 687 O14617 688 O14672 689 O14715690 O14757 691 O14773 692 O14792 693 O14818 694 O14950 695 O15016 696O15021 697 O15040 698 O15075 699 O15117 700 O15145 701 O15212 702 O15372703 O15397 704 O15427 705 O15460 706 O15498 707 O15511 708 O43143 709O43151 710 O43323 711 O43396 712 O43464 713 O43823 714 O43865 715 O60229716 O60279 717 O60281 718 O60423 719 O60456 720 O60500 721 O60518 722O60673 723 O60759 724 O60763 725 O60784 726 O60890 727 O75083 728 O75326729 O75340 730 O75376 731 O75417 732 O75508 733 O75533 734 O75616 735O75718 736 O75792 737 O75937 738 O75954 739 O75962 740 O94855 741 O94973742 O94986 743 O95084 744 O95147 745 O95201 746 O95202 747 O95391 748O95447 749 O95486 750 O95633 751 O95786 752 O95965 753 O95967 754 O95999755 O96019 756 P00367 757 P00491 758 P00505 759 P00558 760 P00918 761P01033 762 P01137 763 P01601 764 P01613 765 P02511 766 P02649 767 P02765768 P02787 769 P02794 770 P03956 771 P04004 772 P04083 773 P04216 774P04424 775 P04632 776 P04899 777 P05114 778 P05121 779 P05166 780 P05387781 P05783 782 P06132 783 P06703 784 P07093 785 P07237 786 P07355 787P07384 788 P07437 789 P07686 790 P07858 791 P07954 792 P08100 793 P08133794 P08195 795 P08476 796 P08590 797 P08758 798 P09211 799 P09238 800P09543 801 P09603 802 P09936 803 P09972 804 P0CG42 805 P0CI25 806 P10124807 P10646 808 P10809 809 P10915 810 P11142 811 P11166 812 P11413 813P11717 814 P11766 815 P11940 816 P12081 817 P13473 818 P13489 819 P13569820 P13646 821 P13674 822 P13716 823 P13987 824 P14174 825 P14209 826P14324 827 P14735 828 P15121 829 P15144 830 P15586 831 P16278 832 P16520833 P16885 834 P17035 835 P17174 836 P17342 837 P17655 838 P17812 839P17931 840 P17936 841 P17980 842 P17987 843 P19113 844 P19623 845 P19823846 P19883 847 P20929 848 P21266 849 P21333 850 P21397 851 P21439 852P21589 853 P21810 854 P21817 855 P21926 856 P21980 857 P22304 858 P22681859 P22897 860 P23276 861 P23284 862 P23396 863 P23470 864 P24043 865P24539 866 P24593 867 P25092 868 P25325 869 P25789 870 P26006 871 P26038872 P26599 873 P26639 874 P26640 875 P27658 876 P28062 877 P30085 878P30530 879 P30622 880 P31937 881 P31939 882 P31948 883 P31949 884 P32926885 P33402 886 P35221 887 P35222 888 P35250 889 P35579 890 P35913 891P35998 892 P36954 893 P37802 894 P40199 895 P40818 896 P40855 897 P40926898 P41091 899 P41221 900 P41252 901 P42336 902 P46063 903 P46821 904P46926 905 P46939 906 P47756 907 P48509 908 P49189 909 P49368 910 P49589911 P49591 912 P49888 913 P50135 914 P50213 915 P50219 916 P50281 917P50395 918 P50591 919 P50990 920 P51397 921 P51610 922 P51813 923 P51948924 P52788 925 P52848 926 P52888 927 P52907 928 P53611 929 P53992 930P54289 931 P54296 932 P54687 933 P54802 934 P55001 935 P55008 936 P55072937 P55083 938 P55268 939 P55285 940 P55287 941 P55884 942 P56159 943P56559 944 P57075 945 P58215 946 P59817 947 P60900 948 P60903 949 P60953950 P60981 951 P61011 952 P61163 953 P61586 954 P61758 955 P61769 956P62140 957 P62166 958 P62195 959 P62249 960 P62280 961 P62328 962 P62333963 P62873 964 P62906 965 P62942 966 P63135 967 P63151 968 P68366 969P68543 970 P69849 971 P78315 972 P78316 973 P78368 974 P78417 975 P78424976 P78504 977 P80303 978 P84077 979 Q00266 980 Q00341 981 Q00587 982Q00796 983 Q01082 984 Q01518 985 Q01650 986 Q01746 987 Q01813 988 Q01973989 Q02045 990 Q02818 991 Q02846 992 Q06033 993 Q06830 994 Q06HC4 995Q07021 996 Q07065 997 Q07343 998 Q07666 999 Q07960 1000 Q08378 1001Q08379 1002 Q08397 1003 Q09328 1004 Q0R2U2 1005 Q0VFZ6 1006 Q108H6 1007Q12830 1008 Q12884 1009 Q12889 1010 Q12907 1011 Q13045 1012 Q13069 1013Q13098 1014 Q13155 1015 Q13283 1016 Q13347 1017 Q13362 1018 Q13416 1019Q13443 1020 Q13464 1021 Q13478 1022 Q13564 1023 Q13591 1024 Q13596 1025Q13682 1026 Q13823 1027 Q13939 1028 Q14055 1029 Q14108 1030 Q14118 1031Q14126 1032 Q14197 1033 Q14257 1034 Q14442 1035 Q14444 1036 Q14523 1037Q14651 1038 Q14683 1039 Q14689 1040 Q14699 1041 Q14764 1042 Q14767 1043Q14839 1044 Q14868 1045 Q14CZ0 1046 Q15008 1047 Q15019 1048 Q15024 1049Q15029 1050 Q15136 1051 Q15228 1052 Q15256 1053 Q15262 1054 Q15413 1055Q15424 1056 Q15464 1057 Q15465 1058 Q15526 1059 Q15596 1060 Q15648 1061Q15753 1062 Q15904 1063 Q16181 1064 Q16204 1065 Q16270 1066 Q16531 1067Q16576 1068 Q16832 1069 Q1KMD3 1070 Q1WIS0 1071 Q24JU4 1072 Q2KHR2 1073Q2LAE2 1074 Q2M2A7 1075 Q2TAZ0 1076 Q2UY09 1077 Q2VIQ3 1078 Q2VIR3 1079Q2VPA4 1080 Q2VPJ0 1081 Q2YDB1 1082 Q32ND0 1083 Q3B7X1 1084 Q3KNR5 1085Q3KNT2 1086 Q3LIF6 1087 Q3SXZ7 1088 Q3ZCM1 1089 Q3ZLR7 1090 Q3ZTS5 1091Q461N2 1092 Q494Z1 1093 Q495C1 1094 Q495F8 1095 Q496J3 1096 Q4G0H8 1097Q4KMZ1 1098 Q4QRJ3 1099 Q4VAN1 1100 Q4VY13 1101 Q502X7 1102 Q504U6 1103Q53EL0 1104 Q53F80 1105 Q53FN7 1106 Q53FW8 1107 Q53H08 1108 Q53H26 1109Q53HA9 1110 Q53QV8 1111 Q53SB6 1112 Q53XL7 1113 Q53Y83 1114 Q53Y86 1115Q53YD7 1116 Q53ZX7 1117 Q540U8 1118 Q548T7 1119 Q549N7 1120 Q54A51 1121Q569L6 1122 Q56N19 1123 Q587J7 1124 Q58F26 1125 Q59ED9 1126 Q59EU4 1127Q59F19 1128 Q59FL3 1129 Q59GL0 1130 Q5BJE1 1131 Q5CZ70 1132 Q5CZA0 1133Q5CZC0 1134 Q5EBM7 1135 Q5HYB0 1136 Q5HYG7 1137 Q5JC44 1138 Q5JPA7 1139Q5JRW7 1140 Q5JTN6 1141 Q5JTW6 1142 Q5JUQ2 1143 Q5JWF2 1144 Q5JXX7 1145Q5JZ08 1146 Q5M8T4 1147 Q5PY49 1148 Q5QJU3 1149 Q5STV5 1150 Q5T0U0 1151Q5T160 1152 Q5T427 1153 Q5T655 1154 Q5T6L9 1155 Q5T7B3 1156 Q5T7W0 1157Q5T9L3 1158 Q5TB52 1159 Q5TCJ2 1160 Q5TCX8 1161 Q5TEZ5 1162 Q5TGS1 1163Q5TH69 1164 Q5U0F2 1165 Q5VT97 1166 Q5VV63 1167 Q5VWT5 1168 Q5VYK3 1169Q5VZK9 1170 Q5VZV1 1171 Q63HP0 1172 Q643R0 1173 Q658R0 1174 Q659E8 1175Q659F6 1176 Q659F8 1177 Q68D86 1178 Q68DA7 1179 Q68DM7 1180 Q68DN3 1181Q68DS2 1182 Q68DS3 1183 Q6AZY7 1184 Q6FGL8 1185 Q6FI91 1186 Q6IAT7 1187Q6IAX5 1188 Q6IB29 1189 Q6IBR6 1190 Q6ICD2 1191 Q6IE37 1192 Q6IPS9 1193Q6K0P9 1194 Q6MZF2 1195 Q6MZM3 1196 Q6NSC9 1197 Q6NTE8 1198 Q6NV74 1199Q6NX68 1200 Q6NZ83 1201 Q6P0Q8 1202 Q6P2D0 1203 Q6P3W7 1204 Q6P528 1205Q6P5Z4 1206 Q6PCE3 1207 Q6PEW0 1208 Q6PIK3 1209 Q6PIW4 1210 Q6PJ36 1211Q6PKF2 1212 Q6Q4G3 1213 Q6Q759 1214 Q6UWP2 1215 Q6UWW8 1216 Q6UXE8 1217Q6UXH8 1218 Q6WCQ1 1219 Q6WRI0 1220 Q6XZE5 1221 Q6YNQ1 1222 Q6ZMW9 1223Q6ZN06 1224 Q6ZN13 1225 Q6ZNK6 1226 Q6ZNS6 1227 Q6ZPB4 1228 Q6ZR08 1229Q6ZR62 1230 Q6ZR81 1231 Q6ZRR7 1232 Q6ZRR9 1233 Q6ZS30 1234 Q6ZSA3 1235Q6ZUX3 1236 Q6ZVH7 1237 Q6ZVJ0 1238 Q6ZWD7 1239 Q70Q86 1240 Q76KX8 1241Q76M96 1242 Q76N11 1243 Q7KZF4 1244 Q7L112 1245 Q7L2K0 1246 Q7L5A8 1247Q7L5N1 1248 Q7LBC6 1249 Q7LBX6 1250 Q7LFX5 1251 Q7RTM4 1252 Q7RTU9 1253Q7RTV5 1254 Q7Z304 1255 Q7Z3G2 1256 Q7Z3L3 1257 Q7Z410 1258 Q7Z4H8 1259Q7Z4L4 1260 Q7Z4X1 1261 Q7Z583 1262 Q7Z5L2 1263 Q7Z5M5 1264 Q7Z5X8 1265Q7Z679 1266 Q7Z7G0 1267 Q85ZX8 1268 Q86SG6 1269 Q86SJ2 1270 Q86T20 1271Q86TS1 1272 Q86U45 1273 Q86U53 1274 Q86UB2 1275 Q86UE9 1276 Q86VS8 1277Q86W50 1278 Q86W67 1279 Q86WT6 1280 Q86XA9 1281 Q86YW9 1282 Q8IU62 1283Q8IUU5 1284 Q8IUX7 1285 Q8IVB1 1286 Q8IVL5 1287 Q8IW47 1288 Q8IWB6 1289Q8IWE2 1290 Q8IYT3 1291 Q8IZF3 1292 Q8IZG2 1293 Q8IZP2 1294 Q8IZZ8 1295Q8N137 1296 Q8N248 1297 Q8N212 1298 Q8N2S1 1299 Q8N412 1300 Q8N414 1301Q8N427 1302 Q8N474 1303 Q8N4E4 1304 Q8N508 1305 Q8N6J3 1306 Q8N7D9 1307Q8N7X0 1308 Q8N822 1309 Q8N8F1 1310 Q8N961 1311 Q8N9D1 1312 Q8NA02 1313Q8NA54 1314 Q8NBJ4 1315 Q8NBS9 1316 Q8NCC3 1317 Q8NDD5 1318 Q8NDM7 1319Q8NE71 1320 Q8NEL0 1321 Q8NEN0 1322 Q8NEP9 1323 Q8NEZ5 1324 Q8NFF3 1325Q8NFW1 1326 Q8NFZ4 1327 Q8NHX6 1328 Q8TB37 1329 Q8TB73 1330 Q8TBC4 1331Q8TBW1 1332 Q8TCE6 1333 Q8TCU5 1334 Q8TCY9 1335 Q8TD47 1336 Q8TDD1 1337Q8TDD2 1338 Q8TDR4 1339 Q8TEC9 1340 Q8TEF5 1341 Q8TF27 1342 Q8TF62 1343Q8WUQ3 1344 Q8WUS6 1345 Q8WV16 1346 Q8WW02 1347 Q8WWH5 1348 Q8WWI5 1349Q8WWN8 1350 Q8WWV3 1351 Q8WXA3 1352 Q8WXG9 1353 Q8WXJ2 1354 Q8WY19 1355Q8WZ74 1356 Q8WZ99 1357 Q8WZA0 1358 Q92545 1359 Q92598 1360 Q92614 1361Q92621 1362 Q92667 1363 Q92673 1364 Q92777 1365 Q92791 1366 Q92794 1367Q92878 1368 Q92896 1369 Q92913 1370 Q93008 1371 Q93009 1372 Q969J2 1373Q969P5 1374 Q969X0 1375 Q96AB3 1376 Q96AC1 1377 Q96AG3 1378 Q96AL5 1379Q96AW4 1380 Q96AY3 1381 Q96B45 1382 Q96B70 1383 Q96C11 1384 Q96C57 1385Q96CP2 1386 Q96CW1 1387 Q96DX8 1388 Q96EB6 1389 Q96ES0 1390 Q96HK3 1391Q96186 1392 Q961C2 1393 Q96119 1394 Q96J40 1395 Q96J66 1396 Q96J72 1397Q96JC1 1398 Q96JH7 1399 Q96JM2 1400 Q96JP2 1401 Q96L42 1402 Q96M20 1403Q96M97 1404 Q96MC4 1405 Q96MY7 1406 Q96N32 1407 Q96N83 1408 Q96NX8 1409Q96Q42 1410 Q96QB0 1411 Q96QF1 1412 Q96RE7 1413 Q96RT7 1414 Q96RZ6 1415Q96T23 1416 Q96T70 1417 Q96TA1 1418 Q96TC7 1419 Q96TG0 1420 Q99422 1421Q99471 1422 Q99497 1423 Q99525 1424 Q99608 1425 Q99613 1426 Q99623 1427Q99626 1428 Q99712 1429 Q99767 1430 Q99868 1431 Q99874 1432 Q9BR76 1433Q9BRM9 1434 Q9BSH8 1435 Q9BSJ5 1436 Q9BSK1 1437 Q9BT66 1438 Q9BTF5 1439Q9BTG3 1440 Q9BTN2 1441 Q9BTZ8 1442 Q9BU99 1443 Q9BUB5 1444 Q9BV90 1445Q9BW52 1446 Q9BW58 1447 Q9BWZ5 1448 Q9BXF1 1449 Q9BXF3 1450 Q9BXL5 1451Q9BYT8 1452 Q9BZ74 1453 Q9BZE3 1454 Q9BZQ4 1455 Q9C040 1456 Q9GZP4 1457Q9GZT8 1458 Q9GZV3 1459 Q9H0A0 1460 Q9H0B8 1461 Q9H0E2 1462 Q9H0H0 1463Q9H013 1464 Q9H0P5 1465 Q9H106 1466 Q9H128 1467 Q9H156 1468 Q9H173 1469Q9H1J1 1470 Q9H1J7 1471 Q9H1Z3 1472 Q9H1Z5 1473 Q9H299 1474 Q9H2J7 1475Q9H2K3 1476 Q9H2P8 1477 Q9H3P9 1478 Q9H3S7 1479 Q9H4A4 1480 Q9H4D0 1481Q9H4F8 1482 Q9H4M9 1483 Q9H515 1484 Q9H5L6 1485 Q9H5R6 1486 Q9H6D4 1487Q9H7D0 1488 Q9H7H0 1489 Q9H7H5 1490 Q9H7P3 1491 Q9H8E8 1492 Q9H8G5 1493Q9H8H3 1494 Q9H8S9 1495 Q9H8V8 1496 Q9H903 1497 Q9H9Q2 1498 Q9HA51 1499Q9HB40 1500 Q9HBH9 1501 Q9HC39 1502 Q9HC87 1503 Q9HCC0 1504 Q9HCF4 1505Q9HCG1 1506 Q9HCM1 1507 Q9HCN6 1508 Q9HD26 1509 Q9NNZ5 1510 Q9NPK3 1511Q9NPW9 1512 Q9NQW6 1513 Q9NR18 1514 Q9NRA1 1515 Q9NRN5 1516 Q9NRY6 1517Q9NS13 1518 Q9NS15 1519 Q9NS87 1520 Q9NSJ3 1521 Q9NTH6 1522 Q9NUY8 1523Q9NVA2 1524 Q9NVE4 1525 Q9NVE5 1526 Q9NVR2 1527 Q9NWL6 1528 Q9NXA5 1529Q9NXC3 1530 Q9NXZ1 1531 Q9NY33 1532 Q9NYM2 1533 Q9NYQ7 1534 Q9NZ08 1535Q9NZN4 1536 Q9NZP2 1537 Q9NZQ9 1538 Q9NZU1 1539 Q9NZV1 1540 Q9P0K1 1541Q9P1S5 1542 Q9P1W9 1543 Q9P246 1544 Q9P2L0 1545 Q9P2W4 1546 Q9P2X3 1547Q9TQL1 1548 Q9UBC0 1549 Q9UBF2 1550 Q9UBP4 1551 Q9UBQ5 1552 Q9UBU7 1553Q9UC56 1554 Q9UCV3 1555 Q9UDG3 1556 Q9UDL5 1557 Q9UEB7 1558 Q9UF52 1559Q9UF61 1560 Q9UFC6 1561 Q9UFT4 1562 Q9UFW3 1563 Q9UGJ1 1564 Q9UHY1 1565Q9U142 1566 Q9UIF8 1567 Q9UIY4 1568 Q9UJ70 1569 Q9UJ71 1570 Q9UJC6 1571Q9UJF2 1572 Q9UJJ2 1573 Q9UJJ9 1574 Q9UJU4 1575 Q9UKA4 1576 Q9UKJ8 1577Q9UL63 1578 Q9UL95 1579 Q9ULA0 1580 Q9ULD6 1581 Q9ULG3 1582 Q9ULJ9 1583Q9ULT8 1584 Q9ULU8 1585 Q9ULY4 1586 Q9UM21 1587 Q9UMG6 1588 Q9UMG7 1589Q9UMN6 1590 Q9UMV8 1591 Q9UN71 1592 Q9UNN5 1593 Q9UNP5 1594 Q9UNS2 1595Q9UP50 1596 Q9UPJ7 1597 Q9UQP3 1598 Q9UQR1 1599 Q9UQR9 1600 Q9Y224 1601Q9Y234 1602 Q9Y240 1603 Q9Y264 1604 Q9Y2B0 1605 Q9Y2E5 1606 Q9Y2G5 1607Q9Y2H8 1608 Q9Y2M5 1609 Q9Y2S0 1610 Q9Y2T6 1611 Q9Y2X7 1612 Q9Y3I1 1613Q9Y3R5 1614 Q9Y3X0 1615 Q9Y423 1616 Q9Y450 1617 Q9Y467 1618 Q9Y4C4 1619Q9Y4E8 1620 Q9Y4G2 1621 Q9Y4G8 1622 Q9Y4M4 1623 Q9Y5E6 1624 Q9Y5S2 1625Q9Y5V3 1626 Q9Y5Z7 1627 Q9Y687 1628 Q9Y6B7 1629 Q9Y6D5 1630 Q9Y6N7 1631Q9Y6V7 1632 R4GNE8 1633 R4GNI9 1634 S4R2Z0 1635 S4R354 1636 S4R3N6 1637S4R3P9 1638 T2FFJ4 1639 U3KQ69 1640 U3KQL2 1641 U5TP13 1642 V9GYP9 1643V9HVU7 1644 V9HW54 1645 V9HWB9 1646 W8JD10 1647 X5D2S9 1648 X5DNF2 1649X6R3I0 1650 X6R772 1651 X6R8W7 1652 X6R922 1653 X6RLN4

Therefore, the disclosed EVs are unique based on the source of cellsfrom which they are derived. Moreover, these proteins can be used as asignature to identify the EVs.

Discussion

This is the first documentation that we are aware of on a scale thatrequires the Amicon stirred-cell ultrafiltration units, allowingfiltration and EV enrichment from 24 liters of media within one week, anamount of media that could not logistically be purified byultracentrifuge, and would require intense manpower and multiplecentrifuges using the smaller Centricon/Amicon centrifugal filter units.

The inclusion of hFGF2 from cell culture media in NPEX™, combined withthe ability of NPEX EVs to distribute to targets within the CNS (asdemonstrated in biodistribution section), suggests that these EVs canpotentially deliver hFGF2 across the blood brain barrier to target CNStissue. These results suggest that a potential method for deliveringlarge molecules including proteins (hFGF2 in the example above) totargets in vivo, including the CNS, using extracellular vesicles (NPEX™by supplementing the EV source cell (hNP1™ in example above) culturemedia with the molecule of interest.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Publications cited herein andthe materials for which they are cited are specifically incorporated byreference.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

What is claimed is:
 1. A method of reducing inflammation in the brain ofa subject, comprising administering to the subject an effective amountof a pharmaceutical composition comprising isolated extracellularvesicles (EVs) derived from non-transformed neural progenitor cells. 2.The method of claim 1, wherein administering the pharmaceuticalcomposition increases the number of regulatory T cells in thecirculation of the subject.
 3. The method of claim 1, whereinadministering the pharmaceutical composition reduces the number of Thelper cells in the circulation of the subject.
 4. The method of claim1, wherein administering the pharmaceutical composition increases thenumber of M2 macrophages in the circulation of the subject.
 5. Themethod of claim 1, wherein administering the pharmaceutical compositionsuppresses an inflammatory response of M1 macrophages in the subject. 6.The method of claim 1, wherein administering the pharmaceuticalcomposition enhances an anti-inflammatory response of M2 macrophages inthe subject.
 7. The method of claim 1, wherein administering thepharmaceutical composition reduces a level if IL-17 in the subject. 8.The method of claim 1, wherein administering the pharmaceuticalcomposition increases a level of IL-10 in the subject.
 9. The method ofclaim 1, wherein the neural progenitor cells are human cells.
 10. Themethod of claim 9, wherein the neural progenitor cells are derived fromhuman pluripotent stem cells.
 11. The method of claim 9, wherein theneural progenitor cells are derived from human induced pluripotent stemcells.
 12. The method of claim 9, wherein the neural progenitor cellsare derived from human embryonic stem cells.
 13. The method of claim 1,wherein the neural progenitor cells express one or more markers selectedfrom the group consisting of nestin, SOX1, and SOX2, and do not expressOCT4.
 14. The method of claim 1, wherein the pharmaceutical compositioncomprises about 1 mg to about 750 mg of EVs.
 15. The method of claim 1,wherein the EVs comprise exosomes.
 16. The method of claim 1, whereinthe EVs are about 20 nm to 10 μm in size.
 17. The method of claim 1,wherein the EVs are about 25 nm to about 250 nm in size.
 18. The methodof claim 1, wherein the pharmaceutical composition is administeredintravenously.
 19. The method of claim 1, wherein the pharmaceuticalcomposition is administered intranasally.
 20. The method of claim 1,wherein the pharmaceutical composition is administered via a route ofdelivery selected from intrathecal, intraperitoneal (IP), oral, ordirect delivery into the brain.
 21. The method of claim 1, wherein theEVs further comprise an siRNA or an antisense oligonucleotide.
 22. Themethod of claim 1, wherein the EVs further comprise an exogenous peptideor an exogenous protein.
 23. The method of claim 1, wherein the EVsfurther comprise a small molecule.
 24. The method of claim 1, whereinthe EVs further comprise an antibody.
 25. The method of claim 1, whereinthe subject has or is at risk for a traumatic brain injury.
 26. Themethod of claim 1, wherein the subject has or is at risk for a spinalcord injury.
 27. The method of claim 1, wherein the subject has or is atrisk for a neurodegenerative disease.
 28. The method of claim 27,wherein the neurodegenerative disease is one or more of Alzheimer'sdisease, Parkinson's disease, a Parkinson's-related disorder,Huntington's disease, amyotrophic lateral sclerosis, prion disease,motor neuron disease (MND), spinocerebellar ataxia (SCA), or spinalmuscular atrophy (SMA).
 29. The method of claim 1, wherein the subjecthas swelling in the brain, and wherein the method reduces the swellingin the brain.